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1
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Martí-Clúa J. 5-Bromo-2'-deoxyuridine labeling: historical perspectives, factors influencing the detection, toxicity, and its implications in the neurogenesis. Neural Regen Res 2024; 19:302-308. [PMID: 37488882 PMCID: PMC10503596 DOI: 10.4103/1673-5374.379038] [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: 03/20/2023] [Revised: 04/25/2023] [Accepted: 05/25/2023] [Indexed: 07/26/2023] Open
Abstract
The halopyrimidine 5-bromo-2'-deoxyuridine (BrdU) is an exogenous marker of DNA synthesis. Since the introduction of monoclonal antibodies against BrdU, an increasing number of methodologies have been used for the immunodetection of this synthesized bromine-tagged base analogue into replicating DNA. BrdU labeling is widely used for identifying neuron precursors and following their fate during the embryonic, perinatal, and adult neurogenesis in a variety of vertebrate species including birds, reptiles, and mammals. Due to BrdU toxicity, its incorporation into replicating DNA presents adverse consequences on the generation, survival, and settled patterns of cells. This may lead to false results and misinterpretation in the identification of proliferative neuroblasts. In this review, I will indicate the detrimental effects of this nucleoside during the development of the central nervous system, as well as the reliability of BrdU labeling to detect proliferating neuroblasts. Moreover, it will show factors influencing BrdU immunodetection and the contribution of this nucleoside to the study of prenatal, perinatal, and adult neurogenesis. Human adult neurogenesis will also be discussed. It is my hope that this review serves as a reference for those researchers who focused on detecting cells that are in the synthetic phase of the cell cycle.
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Affiliation(s)
- Joaquín Martí-Clúa
- Unidad de Citología e Histología. Departament de Biologia Cel·lular, de Fisiologia i d’Immunologia. Facultad de Biociencias. Institut de Neurociències. Universidad Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
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2
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El-Kalyoubi S, El-Sebaey SA, Elfeky SM, AL-Ghulikah HA, El-Zoghbi MS. Novel Aminopyrimidine-2,4-diones, 2-Thiopyrimidine-4-ones, and 6-Arylpteridines as Dual-Target Inhibitors of BRD4/PLK1: Design, Synthesis, Cytotoxicity, and Computational Studies. Pharmaceuticals (Basel) 2023; 16:1303. [PMID: 37765111 PMCID: PMC10535864 DOI: 10.3390/ph16091303] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2023] [Revised: 08/30/2023] [Accepted: 09/11/2023] [Indexed: 09/29/2023] Open
Abstract
Structural-based drug design and solvent-free synthesis were combined to obtain three novel series of 5-arylethylidene-aminopyrimidine-2,4-diones (4, 5a-c, 6a,b), 5-arylethylidene-amino-2-thiopyrimidine-4-ones (7,8), and 6-arylpteridines (9,10) as dual BRD4 and PLK1 inhibitors. MTT assays of synthesized compounds against breast (MDA-MB-231), colorectal (HT-29), and renal (U-937) cancer cells showed excellent-to-good cytotoxic activity, compared to Methotrexate; MDA-MB-231 were the most sensitive cancer cells. The most active compounds were tested against normal Vero cells. Compounds 4 and 7 significantly inhibited BRD4 and PLK1, with IC50 values of 0.029, 0.042 µM, and 0.094, 0.02 µM, respectively, which are nearly comparable to volasertib (IC50 = 0.017 and 0.025 µM). Compound 7 triggered apoptosis and halted cell growth at the G2/M phase, similarly to volasertib. It also upregulated the BAX and caspase-3 markers while downregulating the Bcl-2 gene. Finally, active compounds fitted the volasertib binding site at BRD4 and PLK1 and showed ideal drug-like properties and pharmacokinetics, making them promising anticancer candidates.
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Affiliation(s)
- Samar El-Kalyoubi
- Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Port Said University, Port Said 42511, Egypt
| | - Samiha A. El-Sebaey
- Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Youssef Abbas Street, Cairo 11754, Egypt
| | - Sherin M. Elfeky
- Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 355516, Egypt;
| | - Hanan A. AL-Ghulikah
- Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia;
| | - Mona S. El-Zoghbi
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Menoufia University, Menoufia, Gamal Abd Al-Nasir Street, Shibin-Elkom 32511, Egypt;
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3
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Martí-Clúa J. Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues. BIOLOGY 2023; 12:885. [PMID: 37372169 DOI: 10.3390/biology12060885] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/21/2023] [Revised: 06/12/2023] [Accepted: 06/17/2023] [Indexed: 06/29/2023]
Abstract
Tritiated thymidine autoradiography, 5-bromo-2'-deoxyuridine (BrdU) 5-chloro-2'-deoxyuridine (CldU), 5-iodo-2'-deoxyuridine (IdU), and 5-ethynyl-2'-deoxyiridine (EdU) labeling have been used for identifying the fraction of cells undergoing the S-phase of the cell cycle and to follow the fate of these cells during the embryonic, perinatal, and adult life in several species of vertebrate. In this current review, I will discuss the dosage and times of exposition to the aforementioned thymidine analogues to label most of the cells undergoing the S-phase of the cell cycle. I will also show how to infer, in an asynchronous cell population, the duration of the G1, S, and G2 phases, as well as the growth fraction and the span of the whole cell cycle on the base of some labeling schemes involving a single administration, continuous nucleotide analogue delivery, and double labeling with two thymidine analogues. In this context, the choice of the optimal dose of BrdU, CldU, IdU, and EdU to label S-phase cells is a pivotal aspect to produce neither cytotoxic effects nor alter cell cycle progression. I hope that the information presented in this review can be of use as a reference for researchers involved in the genesis of tissues and organs.
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Affiliation(s)
- Joaquín Martí-Clúa
- Unidad de Citología e Histología, Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
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4
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Ganesan N, Ronsmans S, Hoet P. Methods to Assess Proliferation of Stimulated Human Lymphocytes In Vitro: A Narrative Review. Cells 2023; 12:cells12030386. [PMID: 36766728 PMCID: PMC9913443 DOI: 10.3390/cells12030386] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 12/10/2022] [Accepted: 01/18/2023] [Indexed: 01/24/2023] Open
Abstract
The ability to monitor lymphocyte responses is critical for developing our understanding of the immune response in humans. In the current clinical setting, relying on the metabolic incorporation of [3H] thymidine into cellular DNA via a lymphocyte proliferation test (LPT) is the only method that is routinely performed to determine cell proliferation. However, techniques that measure DNA synthesis with a radioactive material such as [3H] thymidine are intrinsically more sensitive to the different stages of the cell cycle, which could lead to over-analyses and the subsequent inaccurate interpretation of the information provided. With cell proliferation assays, the output should preferably provide a direct and accurate measurement of the number of actively dividing cells, regardless of the stimuli properties or length of exposure. In fact, an ideal technique should have the capacity to measure lymphocyte responses on both a quantitative level, i.e., cumulative magnitude of lymphoproliferative response, and a qualitative level, i.e., phenotypical and functional characterization of stimulated immune cells. There are many LPT alternatives currently available to measure various aspects of cell proliferation. Of the nine techniques discussed, we noted that the majority of these LPT alternatives measure lymphocyte proliferation using flow cytometry. Across some of these alternatives, the covalent labelling of cells with a high fluorescence intensity and low variance with minimal cell toxicity while maximizing the number of detectable cell divisions or magnitude of proliferation was achieved. Herein, we review the performance of these different LPT alternatives and address their compatibility with the [3H] thymidine LPT so as to identify the "best" alternative to the [3H] thymidine LPT.
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Affiliation(s)
- Nirosha Ganesan
- Laboratory of Toxicology, Unit of Environment & Health, Department of Public Health and Primary Care, KU Leuven, 3000 Leuven, Belgium
- Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), KU Leuven, 3000 Leuven, Belgium
| | - Steven Ronsmans
- Laboratory of Toxicology, Unit of Environment & Health, Department of Public Health and Primary Care, KU Leuven, 3000 Leuven, Belgium
- Clinic for Occupational and Environmental Medicine, Department of Respiratory Diseases, University Hospitals Leuven, 3000 Leuven, Belgium
| | - Peter Hoet
- Laboratory of Toxicology, Unit of Environment & Health, Department of Public Health and Primary Care, KU Leuven, 3000 Leuven, Belgium
- Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), KU Leuven, 3000 Leuven, Belgium
- Correspondence:
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5
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Bhuvaragavan S, Sruthi K, Nivetha R, Ramaraj P, Hilda K, Meenakumari M, Janarthanan S. Insect galectin stimulates the human CD4+ T cell proliferation by regulating inflammation (T cell and monocyte) through Th2 immune response. Process Biochem 2022. [DOI: 10.1016/j.procbio.2022.12.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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6
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Janssens Y, Joye J, Waerlop G, Clement F, Leroux-Roels G, Leroux-Roels I. The role of cell-mediated immunity against influenza and its implications for vaccine evaluation. Front Immunol 2022; 13:959379. [PMID: 36052083 PMCID: PMC9424642 DOI: 10.3389/fimmu.2022.959379] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Accepted: 07/27/2022] [Indexed: 12/25/2022] Open
Abstract
Influenza vaccines remain the most effective tools to prevent flu and its complications. Trivalent or quadrivalent inactivated influenza vaccines primarily elicit antibodies towards haemagglutinin and neuraminidase. These vaccines fail to induce high protective efficacy, in particular in older adults and immunocompromised individuals and require annual updates to keep up with evolving influenza strains (antigenic drift). Vaccine efficacy declines when there is a mismatch between its content and circulating strains. Current correlates of protection are merely based on serological parameters determined by haemagglutination inhibition or single radial haemolysis assays. However, there is ample evidence showing that these serological correlates of protection can both over- or underestimate the protective efficacy of influenza vaccines. Next-generation universal influenza vaccines that induce cross-reactive cellular immune responses (CD4+ and/or CD8+ T-cell responses) against conserved epitopes may overcome some of the shortcomings of the current inactivated vaccines by eliciting broader protection that lasts for several influenza seasons and potentially enhances pandemic preparedness. Assessment of cellular immune responses in clinical trials that evaluate the immunogenicity of these new generation vaccines is thus of utmost importance. Moreover, studies are needed to examine whether these cross-reactive cellular immune responses can be considered as new or complementary correlates of protection in the evaluation of traditional and next-generation influenza vaccines. An overview of the assays that can be applied to measure cell-mediated immune responses to influenza with their strengths and weaknesses is provided here.
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Affiliation(s)
- Yorick Janssens
- Center for Vaccinology (CEVAC), Ghent University, Ghent, Belgium
| | - Jasper Joye
- Center for Vaccinology (CEVAC), Ghent University Hospital, Ghent, Belgium
| | - Gwenn Waerlop
- Center for Vaccinology (CEVAC), Ghent University, Ghent, Belgium
| | - Frédéric Clement
- Center for Vaccinology (CEVAC), Ghent University, Ghent, Belgium
| | - Geert Leroux-Roels
- Center for Vaccinology (CEVAC), Ghent University, Ghent, Belgium
- Center for Vaccinology (CEVAC), Ghent University Hospital, Ghent, Belgium
| | - Isabel Leroux-Roels
- Center for Vaccinology (CEVAC), Ghent University, Ghent, Belgium
- Center for Vaccinology (CEVAC), Ghent University Hospital, Ghent, Belgium
- *Correspondence: Isabel Leroux-Roels,
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7
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Monitoring Cellular Proliferation and Apoptosis in Atherosclerosis Plaques and Intimal Thickenings. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2022; 2419:507-519. [PMID: 35237985 DOI: 10.1007/978-1-0716-1924-7_31] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Immunohistochemistry for specific proteins characteristic of proliferative or apoptotic cells allows for monitoring of these cell behaviors in biological tissues samples, including atherosclerotic plaques and intimal thickenings. Proliferating cell nuclear antigen (PCNA) and Ki-67 are widely used markers of cell proliferation and cleaved caspase-3 is a well-established marker of apoptosis that can be detected in tissue samples using immunohistochemistry. This technique enables quantification of the abundance of these proteins and provides information on the distribution of these biomarkers in tissues. By combining with immunohistochemistry for specific cell type markers, it is also possible to determine which cell types are proliferating or undergoing apoptosis. Here, we detail protocols for immunohistochemistry of PCNA, Ki-67, and cleaved caspase-3 for evaluation of cellular proliferation and apoptosis in atherosclerotic plaques in vivo. In addition, we outline methods for the quantification and localization of cell proliferation using bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) and ethynyldeoxyuridine/5-ethynyl-2 ́-deoxyuridine(EdU) labeled tissue samples collected from animals exposed to BrdU or EdU.
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8
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Abstract
5-Bromo-2'-deoxyuridine (bromodeoxyuridine (BrdU)), a modified nucleotide and analog of thymidine, is commonly used for detecting proliferating cells. For detection, an anti-BrdU antibody (probe) with a fluorescent dye is applied to bind the BrdU label after DNA denaturation. In this protocol, we provide the BrdU labeling method for both in vitro and in vivo studies, along with immunocytochemistry (ICC)/immunofluorescence (IF) and immunohistochemistry (IHC) staining procedures, respectively. Multicolor staining is also presented as an option to detect the co-distribution of two or multiple antigens in the same sample, making it possible to visualize the location of different molecules at the same time.
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Affiliation(s)
- Jihang Yu
- Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, ON, Canada
| | - Zhixiang Wang
- Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada
| | - Yi Wang
- Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, ON, Canada.
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.
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9
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Wadey KS, Somos A, Cross SJ, Reolizo LM, Johnson JL, George SJ. Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro. Methods Mol Biol 2022; 2419:133-167. [PMID: 35237963 DOI: 10.1007/978-1-0716-1924-7_9] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.
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Affiliation(s)
- Kerry S Wadey
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | - Alexandros Somos
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | - Stephen J Cross
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | - Lien M Reolizo
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | - Jason L Johnson
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK
| | - Sarah J George
- Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK.
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10
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Wang T, Liao JC, Wang X, Wang QS, Wan KY, Yang YY, He Q, Zhang JX, Chen G, Li W. Unexpected BrdU inhibition on astrocyte-to-neuron conversion. Neural Regen Res 2021; 17:1526-1534. [PMID: 34916438 PMCID: PMC8771121 DOI: 10.4103/1673-5374.325747] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
5-Bromo-2′-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.
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Affiliation(s)
- Tao Wang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Jian-Cheng Liao
- Department of Neurosurgery, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China
| | - Xu Wang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Qing-Song Wang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Kai-Ying Wan
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Yi-Yi Yang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Qing He
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Jia-Xuan Zhang
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Gong Chen
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
| | - Wen Li
- Guangdong-Hong Kong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, Guangdong Province, China
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11
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Rodneva SM, Osipov AA, Guryev DV, Tsishnatti AA, Fedotov YА, Yashkina EI, Vorobyova NY, Maksimov AA, Kochetkov OA, Samoylov AS, Osipov AN. Comparative Study of the γH2AX Foci Forming in Human Lung Fibroblasts Incubated in Media Containing Tritium-Labeled Thymidine or Amino Acids. Bull Exp Biol Med 2021; 172:245-249. [PMID: 34853973 DOI: 10.1007/s10517-021-05370-6] [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: 05/20/2021] [Indexed: 11/30/2022]
Abstract
We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.
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Affiliation(s)
- S M Rodneva
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - A A Osipov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia.,N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - D V Guryev
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia. .,N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, Russia.
| | - A A Tsishnatti
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - Y А Fedotov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia.,N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - E I Yashkina
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - N Y Vorobyova
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia.,N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - A A Maksimov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - O A Kochetkov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - A S Samoylov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia
| | - A N Osipov
- A. I. Burnasyan Federal Medical Biophysical Center, Federal Medical-Biological Agency, Moscow, Russia.,N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, Russia
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12
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Péter B, Boldizsár I, Kovács GM, Erdei A, Bajtay Z, Vörös A, Ramsden JJ, Szabó I, Bősze S, Horvath R. Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation. Biomedicines 2021; 9:1781. [PMID: 34944597 PMCID: PMC8698624 DOI: 10.3390/biomedicines9121781] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 11/19/2021] [Accepted: 11/22/2021] [Indexed: 01/07/2023] Open
Abstract
Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.
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Affiliation(s)
- Beatrix Péter
- Nanobiosensorics Group, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary; (A.V.); (R.H.)
| | - Imre Boldizsár
- Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest, Hungary; (I.B.); (G.M.K.)
- Department of Pharmacognosy, Semmelweis University, Üllői út 26, 1085 Budapest, Hungary
| | - Gábor M. Kovács
- Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest, Hungary; (I.B.); (G.M.K.)
- Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary
| | - Anna Erdei
- Department of Immunology, Eötvös Loránd University, 1117 Budapest, Hungary; (A.E.); (Z.B.)
- MTA-ELTE Immunology Research Group, Eötvös Loránd Research Network (ELKH), Eötvös Loránd University, 1117 Budapest, Hungary
| | - Zsuzsa Bajtay
- Department of Immunology, Eötvös Loránd University, 1117 Budapest, Hungary; (A.E.); (Z.B.)
- MTA-ELTE Immunology Research Group, Eötvös Loránd Research Network (ELKH), Eötvös Loránd University, 1117 Budapest, Hungary
| | - Alexandra Vörös
- Nanobiosensorics Group, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary; (A.V.); (R.H.)
| | - Jeremy J. Ramsden
- Clore Laboratory, University of Buckingham, Buckingham MK18 1EG, UK;
| | - Ildikó Szabó
- MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary; (I.S.); (S.B.)
- National Public Health Center, Albert Flórián út 2-6, 1097 Budapest, Hungary
| | - Szilvia Bősze
- MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary; (I.S.); (S.B.)
- National Public Health Center, Albert Flórián út 2-6, 1097 Budapest, Hungary
| | - Robert Horvath
- Nanobiosensorics Group, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary; (A.V.); (R.H.)
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13
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Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview. J Biol Chem 2021; 297:101345. [PMID: 34717955 PMCID: PMC8592869 DOI: 10.1016/j.jbc.2021.101345] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 10/18/2021] [Accepted: 10/19/2021] [Indexed: 01/14/2023] Open
Abstract
Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.
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El Khoudary SR, Fabio A, Yester JW, Steinhauser ML, Christopher AB, Gyngard F, Adams PS, Morell VO, Viegas M, Da Silva JP, Da Silva LF, Castro-Medina M, McCormick A, Reyes-Múgica M, Barlas M, Liu H, Thomas D, Ammanamanchi N, Sada R, Cuda M, Hartigan E, Groscost DK, Kühn B. Design and rationale of a clinical trial to increase cardiomyocyte division in infants with tetralogy of Fallot. Int J Cardiol 2021; 339:36-42. [PMID: 34265312 DOI: 10.1016/j.ijcard.2021.07.020] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 07/02/2021] [Accepted: 07/08/2021] [Indexed: 12/20/2022]
Abstract
BACKGROUND Patients with Tetralogy of Fallot with pulmonary stenosis (ToF/PS), the most common form of cyanotic congenital heart disease (CHD), develop adverse right ventricular (RV) remodeling, leading to late heart failure and arrhythmia. We recently demonstrated that overactive β-adrenergic receptor signaling inhibits cardiomyocyte division in ToF/PS infants, providing a conceptual basis for the hypothesis that treatment with the β-adrenergic receptor blocker, propranolol, early in life would increase cardiomyocyte division. No data are available in ToF/PS infants on the efficacy of propranolol as a possible novel therapeutic option to increase cardiomyocyte division and potentially reduce adverse RV remodeling. METHODS Using a randomized, double-blind, placebo-controlled trial, we will evaluate the effect of propranolol administration on reactivating cardiomyocyte proliferation to prevent adverse RV remodeling in 40 infants with ToF/PS. Propranolol administration (1 mg/kg po QID) will begin at 1 month of age and last until surgical repair. The primary endpoint is cardiomyocyte division, quantified after 15N-thymidine administration with Multi-isotope Imaging Mass Spectrometry (MIMS) analysis of resected myocardial specimens. The secondary endpoints are changes in RV myocardial and cardiomyocyte hypertrophy. CONCLUSION This trial will be the first study in humans to assess whether cardiomyocyte proliferation can be pharmacologically increased. If successful, the results could introduce a paradigm shift in the management of patients with ToF/PS from a purely surgical approach, to synergistic medical and surgical management. It will provide the basis for future multi-center randomized controlled trials of propranolol administration in infants with ToF/PS and other types of CHD with RV hypertension. CLINICAL TRIAL REGISTRATION The trial protocol was registered at clinicaltrials.gov (NCT04713657).
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Affiliation(s)
- Samar R El Khoudary
- Epidemiology Data Center, Graduate School of Public Health, University of Pittsburgh
| | - Anthony Fabio
- Epidemiology Data Center, Graduate School of Public Health, University of Pittsburgh
| | - Jessie W Yester
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA; Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Matthew L Steinhauser
- Aging Institute, University of Pittsburgh, Bridgeside Point 1, 5th Floor, 100 Technology Drive, Pittsburgh, PA 15219, USA; UPMC Heart and Vascular Institute, UPMC Presbyterian, 200 Lothrop St., Pittsburgh, PA 15213, USA
| | - Adam B Christopher
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Frank Gyngard
- Center for NanoImaging, Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 65 Landsdowne St, Rm 535, Cambridge, MA 02139, USA
| | - Phillip S Adams
- Department of Anesthesiology and Perioperative Medicine, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Victor O Morell
- Pediatric Cardiothoracic Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Melita Viegas
- Pediatric Cardiothoracic Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Jose P Da Silva
- Pediatric Cardiothoracic Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Luciana F Da Silva
- Pediatric Cardiothoracic Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Mario Castro-Medina
- Pediatric Cardiothoracic Surgery, UPMC Children's Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Andrew McCormick
- Vascular Anomaly Center, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Miguel Reyes-Múgica
- Division of Pediatric Pathology, UPMC Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA
| | - Michelle Barlas
- Investigational Drug Service, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Honghai Liu
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA; Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Dawn Thomas
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Niyatie Ammanamanchi
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA; Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Rachel Sada
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Megan Cuda
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Elizabeth Hartigan
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - David K Groscost
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA
| | - Bernhard Kühn
- Division of Cardiology, UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA; Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, 4401 Penn Ave, Pittsburgh, PA 15224, USA; McGowan Institute of Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
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15
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Martí-Clúa J. Incorporation of 5-Bromo-2'-deoxyuridine into DNA and Proliferative Behavior of Cerebellar Neuroblasts: All That Glitters Is Not Gold. Cells 2021; 10:cells10061453. [PMID: 34200598 PMCID: PMC8229392 DOI: 10.3390/cells10061453] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Revised: 06/06/2021] [Accepted: 06/07/2021] [Indexed: 12/27/2022] Open
Abstract
The synthetic halogenated pyrimidine analog, 5-bromo-2'-deoxyuridine (BrdU), is a marker of DNA synthesis. This exogenous nucleoside has generated important insights into the cellular mechanisms of the central nervous system development in a variety of animals including insects, birds, and mammals. Despite this, the detrimental effects of the incorporation of BrdU into DNA on proliferation and viability of different types of cells has been frequently neglected. This review will summarize and present the effects of a pulse of BrdU, at doses ranging from 25 to 300 µg/g, or repeated injections. The latter, following the method of the progressively delayed labeling comprehensive procedure. The prenatal and perinatal development of the cerebellum are studied. These current data have implications for the interpretation of the results obtained by this marker as an index of the generation, migration, and settled pattern of neurons in the developing central nervous system. Caution should be exercised when interpreting the results obtained using BrdU. This is particularly important when high or repeated doses of this agent are injected. I hope that this review sheds light on the effects of this toxic maker. It may be used as a reference for toxicologists and neurobiologists given the broad use of 5-bromo-2'-deoxyuridine to label dividing cells.
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Affiliation(s)
- Joaquín Martí-Clúa
- Unidad de Citología e Histología, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
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16
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Yester JW, Liu H, Gyngard F, Ammanamanchi N, Little KC, Thomas D, Sullivan MLG, Lal S, Steinhauser ML, Kühn B. Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division. Nat Protoc 2021; 16:1995-2022. [PMID: 33627842 PMCID: PMC8221415 DOI: 10.1038/s41596-020-00477-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 11/30/2020] [Indexed: 12/26/2022]
Abstract
Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3-8 months, which is dependent on the timing of surgical repair, and 3-4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.
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Affiliation(s)
- Jessie W Yester
- Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
| | - Honghai Liu
- Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
| | - Frank Gyngard
- Center for NanoImaging, Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA
| | - Niyatie Ammanamanchi
- Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
| | - Kathryn C Little
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
- UPMC Shadyside Hospital, Pittsburgh, PA, USA
| | - Dawn Thomas
- Clinical Research Support Services (CRSS), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
| | - Mara L G Sullivan
- Center for Biologic Imaging, University of Pittsburgh School of Medicine, Department of Cell Biology, Pittsburgh, PA, USA
| | - Sean Lal
- Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA
- Center for NanoImaging, Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA
- School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia
- Division of Cardiology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia
| | - Matthew L Steinhauser
- Center for NanoImaging, Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA.
- UPMC Heart and Vascular Institute, UPMC Presbyterian, Pittsburgh, PA, USA.
- Aging Institute, University of Pittsburgh, Bridgeside Point 1, Pittsburgh, PA, USA.
| | - Bernhard Kühn
- Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh and Department of Pediatrics, Pittsburgh, PA, USA.
- McGowan Institute of Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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17
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Martí-Clúa J. Developmental timetables and gradients of neurogenesis in cerebellar Purkinje cells and deep glutamatergic neurons: A comparative study between the mouse and the rat. Anat Rec (Hoboken) 2021; 304:2856-2864. [PMID: 33620144 DOI: 10.1002/ar.24607] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2020] [Revised: 10/31/2020] [Accepted: 11/11/2020] [Indexed: 12/20/2022]
Abstract
The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([3 H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [3 H]TdR on embryonic days (E) 11-12, E12-13, E13-14, E14-15, E15-16, and E16-17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.
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Affiliation(s)
- Joaquín Martí-Clúa
- Unidad de Citología e Histología. Departament de Biologia Cel·lular, de Fisiologia i d'Immunologia. Facultad de Biociencias, Institut de Neurociències. Universidad Autónoma de Barcelona, Bellaterra, Barcelona, Spain
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18
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Zhang D, Liu C, Li H, Jiao J. Deficiency of STING Signaling in Embryonic Cerebral Cortex Leads to Neurogenic Abnormalities and Autistic-Like Behaviors. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2020; 7:2002117. [PMID: 33304758 PMCID: PMC7710002 DOI: 10.1002/advs.202002117] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 09/14/2020] [Indexed: 05/27/2023]
Abstract
STING is known as a central adaptor for sensing cytosolic DNA sensing. Recent studies have provided evidence that STING response is divergent among different cell types. Here, this work demonstrates that STING controls neural progenitor cells (NPCs) by sensing DNA damage in NPCs. The deletion of STING reduces neuronal differentiation and increases proliferation of mouse and human NPCs. Furthermore, STINGcKO mice display autistic-like behaviors. In NPCs, STING specifically recruits IKKβ and activates nuclear factor κB (NF-κB) through phosphorylation. NF-κB binds to ALX4 promoter and triggers ALX4 transcription. In addition, tumor necrosis factor α, an activator of NF-κB, can rescue some phenotypes caused by STING deletion in mice. Together, the findings show that STING signaling is essential for neuronal gene expression program and has profound consequences on brain function.
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Affiliation(s)
- Dongming Zhang
- State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijing100101China
- Medical SchoolUniversity of Chinese Academy of SciencesBeijing100049China
| | - Chang Liu
- Academy for Advanced Interdisciplinary StudiesPeking UniversityBeijing100871China
| | - Hong Li
- State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijing100101China
| | - Jianwei Jiao
- State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijing100101China
- Medical SchoolUniversity of Chinese Academy of SciencesBeijing100049China
- Innovation Academy for Stem Cell and RegenerationBeijing100101China
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19
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Pantazi P, Carollo E, Carter DRF, Brooks SA. A practical toolkit to study aspects of the metastatic cascade in vitro. Acta Histochem 2020; 122:151654. [PMID: 33157489 DOI: 10.1016/j.acthis.2020.151654] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 10/23/2020] [Accepted: 10/28/2020] [Indexed: 12/30/2022]
Abstract
While metastasis - the spread of cancer from the primary location to distant sites in the body - remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a 'scratch' or 'wound-healing' assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes.
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20
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Rodríguez-Vázquez L, Martí J. Administration of 5-bromo-2'-deoxyuridine interferes with neuroblast proliferation and promotes apoptotic cell death in the rat cerebellar neuroepithelium. J Comp Neurol 2020; 529:1081-1096. [PMID: 32785933 DOI: 10.1002/cne.25005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Revised: 07/09/2020] [Accepted: 08/05/2020] [Indexed: 12/13/2022]
Abstract
The current study was conducted to assess whether a single administration of 5-bromo-2'-deoxyuridine (BrdU) interferes with cell proliferation and leads to the activation of apoptotic cellular events in the prenatal cerebellum. BrdU effects across a wide range of doses (25-300 μg/g b.w.) were analyzed using immunohistochemical and ultrastructural procedures. The pregnant rats were injected with BrdU at embryonic day 13, and their fetuses were sacrificed from 5 to 35 hr after exposure. The quantification of several parameters such as the density of mitotic figures, and BrdU and proliferating cell nuclear antigen (PCNA)-reactive cells showed that, in comparison with the saline injected rats, the administration of BrdU impairs the proliferative behavior of neuroepithelial cells. The above-mentioned parameters were significantly reduced in rats injected with 100 μg/g b.w. of BrdU. The reduction was more evident using 200 μg/g b.w. The most severe effects were found with 300 μg/g b.w. of BrdU. The present findings also revealed that high doses of BrdU lead to the activation of apoptotic cellular events as evidenced by both terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for active caspase-3. In comparison with saline rats, many apoptotic cells were found in rats injected with 100 μg/g b.w. of BrdU. The number of dying cells increased with 200 μg/g b.w. The most important number of apoptotic cells were observed in animals injected with 300 μg/g b.w. of BrdU. Ultrastructural studies confirmed the presence of neuroblasts at different stages of apoptosis.
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Affiliation(s)
- Lucía Rodríguez-Vázquez
- Unidad de Citología e Histología, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona, Barcelona, Spain
| | - Joaquín Martí
- Unidad de Citología e Histología, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona, Barcelona, Spain
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21
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Martí J, Rodríguez-Vázquez L. An immunocytochemical approach to the analysis of the cell division cycle in the rat cerebellar neuroepithelium. Cell Cycle 2020; 19:2451-2459. [PMID: 32835583 DOI: 10.1080/15384101.2020.1806425] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Cerebellar neurons are generated from the rhombic lip and the neuroepithelium. In this study, we analyze the histogenesis of the cerebellar neuroepithelium in terms of cellular kinetics. The experimental animals are the offspring of pregnant dams injected with 5-bromo-2'-deoxyuridine (BrdU) on embryonic day 13. We infer the fraction of S-phase cells by examining a range of survival times after a single BrdU-exposure and a cumulative BrdU-labeling sequence, which allow for the derivation of cell-cycle parameters and phase durations. The current results indicate that the dose of BrdU employed (35 mg/kg) provides saturation S-phase labeling from at least 1 h after marker delivery. The duration of G2, mitotic phase, and G1 are 1.2, 0.5, and 6.9 h, respectively. The duration for the S-phase, growth fraction, and the whole cycle are obtained on the basis of two proliferative models, steady-state and exponential growth. Both models provided similar results. In conclusion, our results indicate that the steady-state and the cumulative S-phase labeling paradigms can be adopted to analyze cell cycle parameters in the cerebellar neuroepithelium. Current results can help in understanding the regulatory mechanisms of cerebellar histogenesis and the cell biological mechanisms of the proliferative cycle of the neuroepithelium.
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Affiliation(s)
- Joaquín Martí
- Unidad de Citología e Histología, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona , Barcelona, Spain
| | - Lucía Rodríguez-Vázquez
- Unidad de Citología e Histología, Departament de Biologia Cellular, de Fisiologia i d'Immunologia, Facultad de Biociencias, Institut de Neurociències, Universidad Autónoma de Barcelona , Barcelona, Spain
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22
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Sun L, Fleetwood-Walker S, Mitchell R, Joosten EA, Cheung CW. Prolonged Analgesia by Spinal Cord Stimulation Following a Spinal Injury Associated With Activation of Adult Neural Progenitors. Pain Pract 2020; 20:859-877. [PMID: 32474998 DOI: 10.1111/papr.12921] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2019] [Revised: 01/29/2020] [Accepted: 05/20/2020] [Indexed: 02/06/2023]
Abstract
OBJECTIVES Responses of spinal progenitors to spinal cord stimulation (SCS) following spinal cord injury (SCI) in rats were assessed to reveal their potential contribution to SCS-induced analgesia. METHODS Spinal epidural electrodes were implanted in rats at T12 rostral to a quadrant dorsal horn injury at T13. Further groups additionally received either a microlesion to the dorsolateral funiculus (DLF) or gabapentin (10 mg/kg). SCS was performed at 25 Hz for 10 minutes on day 4 (early SCS) and at 10 Hz for 10 minutes on day 8 (late SCS) after injury. Paw withdrawal threshold (PWT) was measured before injury, 30 minutes before or after SCS, and before cull on day 14, followed by immunostaining assessment. RESULTS Paw withdrawal thresholds in uninjured animals (51.0 ± 4.0 g) were markedly reduced after SCI (17.3 ± 2.2 g). This was significantly increased by early SCS (38.5 ± 5.2 g, P < 0.01) and further enhanced by late SCS (50.9 ± 1.9 g, P < 0.01) over 6 days. Numbers of neural progenitors expressing nestin, Sox2, and doublecortin (DCX) in the spinal dorsal horn were increased 6 days after SCS by 6-fold, 2-fold, and 2.5-fold, respectively (P < 0.05 to 0.01). The elevated PWT evoked by SCS was abolished by DLF microlesions (48.9 ± 2.6 g vs. 19.0 ± 3.9 g, P < 0.01) and the number of nestin-positive cells was reduced to the level without SCS (P < 0.05). Gabapentin enhanced late SCS-induced analgesia from 37.0 ± 3.9 g to 54.0 ± 0.8 g (P < 0.01) and increased gamma-aminobutyric acid (GABA)-ergic neuronal marker vesicular GABA transporter-positive newborn cells 2-fold (P < 0.01). CONCLUSIONS Spinal progenitor cells appear to be activated by SCS via descending pathways, which may be enhanced by gabapentin and potentially contributes to relief of SCI-induced neuropathic pain.
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Affiliation(s)
- Liting Sun
- Brain and Spinal Cord Innovation Research Center, The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, Shanghai, China
| | - Sue Fleetwood-Walker
- Centre for Discovery Brain Sciences, Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh, UK
| | - Rory Mitchell
- Centre for Discovery Brain Sciences, Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh, UK
| | - Elbert A Joosten
- Department of Anesthesiology/Pain Management, Maastricht University Medical Center, Maastricht, The Netherlands
| | - Chi Wai Cheung
- Laboratory and Clinical Research Institute for Pain, Department of Anaesthesiology, University of Hong Kong, HKSAR, China
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23
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PRDM16 orchestrates angiogenesis via neural differentiation in the developing brain. Cell Death Differ 2020; 27:2313-2329. [PMID: 32015502 DOI: 10.1038/s41418-020-0504-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Revised: 01/17/2020] [Accepted: 01/21/2020] [Indexed: 02/07/2023] Open
Abstract
Angiogenesis plays crucial roles in maintaining the complex operation of central nervous system (CNS) development. The architecture of communication between neurogenesis and angiogenesis is essential to maintain normal brain development and function. Hence, any disruption of neuron-vascular communications may lead to the pathophysiology of cerebrovascular diseases and blood-brain barrier (BBB) dysfunction. Here we demonstrate that neural differentiation and communication are required for vascular development. Regarding the cellular and molecular mechanism, our results show that PRDM16 activity determines the production of mature neurons and their specific positions in the neocortex. In the cortical plate (CP), aberrant neurons fail to secrete modular calcium-binding protein 1 (SMOC1), an important neuronal signal that participates in neurovascular communication to regulate CNS angiogenesis. Neuronal SMOC1 interacts with TGFBR1 by activating the transcription factors phospho-Smad2/3 to convey intercellular signals to endothelial cells (ECs) in the TGF-β-Smad signaling pathway. Together, our results highlight a crucial coordinated neurovascular development process orchestrated by PRDM16 and reveal the importance of intimate communication for building the neurovascular network during brain development.
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24
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Rewarding deep brain stimulation at the medial forebrain bundle favours avoidance conditioned response in a remote memory test, hinders extinction and increases neurogenesis. Behav Brain Res 2020; 378:112308. [PMID: 31629001 DOI: 10.1016/j.bbr.2019.112308] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 10/15/2019] [Accepted: 10/15/2019] [Indexed: 12/16/2022]
Abstract
Intracranial Self-Stimulation (ICSS) at the medial forebrain bundle consistently facilitates learning and memory in rats when administered post-training or when administered non-concurrent to training, but its scope regarding remote memory has not yet been studied. The present work aims to test whether the combination of these two forms of ICSS administration can cause a greater persistence of the facilitating effect on remote retention and affect neurogenesis in the dentate gyrus (DG) of the hippocampus. Rats were trained in active avoidance conditioning and tested in two retention sessions (10 and 90 days) and later extinction. Subjects received an ICSS session after each of the five avoidance acquisition sessions (post-training treatment) and half of them also received ten additional ICSS sessions during the rest period between retention tests (non-concurrent treatment). All the stimulated groups showed a higher performance in acquisition and retention sessions, but only the rats receiving both ICSS treatments showed greater resistance to extinction. Remarkably, at seven months, rats receiving the non-concurrent ICSS treatment had a greater number of DCX-positive cells in the DG as well as a higher amount of new-born cells within the granular layer compared to rats that did not receive this additional ICSS treatment. Our present findings significantly extend the temporal window of the facilitating effect of ICSS on active avoidance and demonstrate a neurogenic effect of rewarding medial forebrain bundle stimulation.
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25
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Growth Hormone Promotes Motor Function after Experimental Stroke and Enhances Recovery-Promoting Mechanisms within the Peri-Infarct Area. Int J Mol Sci 2020; 21:ijms21020606. [PMID: 31963456 PMCID: PMC7013985 DOI: 10.3390/ijms21020606] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 01/15/2020] [Accepted: 01/15/2020] [Indexed: 12/31/2022] Open
Abstract
Motor impairment is the most common and widely recognised clinical outcome after stroke. Current clinical practice in stroke rehabilitation focuses mainly on physical therapy, with no pharmacological intervention approved to facilitate functional recovery. Several studies have documented positive effects of growth hormone (GH) on cognitive function after stroke, but surprisingly, the effects on motor function remain unclear. In this study, photothrombotic occlusion targeting the motor and sensory cortex was induced in adult male mice. Two days post-stroke, mice were administered with recombinant human GH or saline, continuing for 28 days, followed by evaluation of motor function. Three days after initiation of the treatment, bromodeoxyuridine was administered for subsequent assessment of cell proliferation. Known neurorestorative processes within the peri-infarct area were evaluated by histological and biochemical analyses at 30 days post-stroke. This study demonstrated that GH treatment improves motor function after stroke by 50%–60%, as assessed using the cylinder and grid walk tests. Furthermore, the observed functional improvements occurred in parallel with a reduction in brain tissue loss, as well as increased cell proliferation, neurogenesis, increased synaptic plasticity and angiogenesis within the peri-infarct area. These findings provide new evidence about the potential therapeutic effects of GH in stroke recovery.
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26
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Li Y, Jiao J. Deficiency of TRPM2 leads to embryonic neurogenesis defects in hyperthermia. SCIENCE ADVANCES 2020; 6:eaay6350. [PMID: 31911949 PMCID: PMC6938698 DOI: 10.1126/sciadv.aay6350] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/05/2019] [Accepted: 11/04/2019] [Indexed: 05/05/2023]
Abstract
Temperature homeostasis is critical for fetal development. The heat sensor protein TRPM2 (transient receptor potential channel M2) plays crucial roles in the heat response, but its function and specific mechanism in brain development remain largely unclear. Here, we observe that TRPM2 is expressed in neural stem cells. In hyperthermia, TRPM2 knockdown and knockout reduce the proliferation of neural progenitor cells (NPCs) and, accordingly, increase premature cortical neuron differentiation. In terms of the mechanism, TRPM2 regulates neural progenitor self-renewal by targeting SP5 (specificity protein 5) via inhibiting the phosphorylation of β-catenin and increasing β-catenin expression. Furthermore, the constitutive expression of TRPM2 or SP5 partly rescues defective NPC proliferation in the TRPM2-deficient embryonic brain. Together, the data suggest that TRPM2 has a critical function in maintaining the NPC pool during heat stress, and the findings provide a framework for understanding how the disruption of the TRPM2 gene may contribute to neurological disorders.
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Affiliation(s)
- Yanxin Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jianwei Jiao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Co-Innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Corresponding author.
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27
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Halim AB. Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays. Curr Drug Discov Technol 2020; 17:2-22. [PMID: 30251606 DOI: 10.2174/1570163815666180925095433] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Revised: 09/13/2018] [Accepted: 09/18/2018] [Indexed: 06/08/2023]
Abstract
Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.
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Affiliation(s)
- Abdel-Baset Halim
- VP Translational Medicine, Biomarkers & Diagnostics, Celldex Therapeutics, 53 Frontage Road, Suite 220, Hampton, NJ 08827-4032, United States
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28
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Islam F, Gopalan V, Lam AK. In Vitro Assays of Biological Aggressiveness of Esophageal Squamous Cell Carcinoma. Methods Mol Biol 2020; 2129:161-175. [PMID: 32056177 DOI: 10.1007/978-1-0716-0377-2_13] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Researchers are developing new techniques and technologies to determine the characteristic features for cancer progression, thereby identifying potential targets and therapeutics to interfere these hallmark processes of cancer pathogenesis. The transformative researches using these in vitro methods have enable researchers to design precision treatments of patients with esophageal squamous cell carcinoma (ESCC). These in vitro methods mainly include analysis of cell proliferation, cytotoxicity, colony formation, invasion, and migration in ESCC cells for analyzing manipulations affecting the biological behavior of ESCC. Because of these studies, important information on molecular mechanisms of different genes and proteins as well as result of therapeutic interventions are confirmed in ESCC.
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Affiliation(s)
- Farhadul Islam
- Cancer Molecular Pathology, School of Medicine, Griffith University, Gold Coast, Queensland, Australia
- Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh
| | - Vinod Gopalan
- Cancer Molecular Pathology, School of Medicine, Griffith University, Gold Coast, Queensland, Australia
| | - Alfred K Lam
- Cancer Molecular Pathology, School of Medicine, Griffith University, Gold Coast, Queensland, Australia.
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29
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Zimmermann T, Maroso M, Beer A, Baddenhausen S, Ludewig S, Fan W, Vennin C, Loch S, Berninger B, Hofmann C, Korte M, Soltesz I, Lutz B, Leschik J. Neural stem cell lineage-specific cannabinoid type-1 receptor regulates neurogenesis and plasticity in the adult mouse hippocampus. Cereb Cortex 2019; 28:4454-4471. [PMID: 30307491 PMCID: PMC6215469 DOI: 10.1093/cercor/bhy258] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Indexed: 12/19/2022] Open
Abstract
Neural stem cells (NSCs) in the adult mouse hippocampus occur in a specific neurogenic niche, where a multitude of extracellular signaling molecules converges to regulate NSC proliferation as well as fate and functional integration. However, the underlying mechanisms how NSCs react to extrinsic signals and convert them to intracellular responses still remains elusive. NSCs contain a functional endocannabinoid system, including the cannabinoid type-1 receptor (CB1). To decipher whether CB1 regulates adult neurogenesis directly or indirectly in vivo, we performed NSC-specific conditional inactivation of CB1 by using triple-transgenic mice. Here, we show that lack of CB1 in NSCs is sufficient to decrease proliferation of the stem cell pool, which consequently leads to a reduction in the number of newborn neurons. Furthermore, neuronal differentiation was compromised at the level of dendritic maturation pointing towards a postsynaptic role of CB1 in vivo. Deteriorated neurogenesis in NSC-specific CB1 knock-outs additionally resulted in reduced long-term potentiation in the hippocampal formation. The observed cellular and physiological alterations led to decreased short-term spatial memory and increased depression-like behavior. These results demonstrate that CB1 expressed in NSCs and their progeny controls neurogenesis in adult mice to regulate the NSC stem cell pool, dendritic morphology, activity-dependent plasticity, and behavior.
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Affiliation(s)
- Tina Zimmermann
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Mattia Maroso
- Department of Neurosurgery, Stanford University, USA
| | - Annika Beer
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Sarah Baddenhausen
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Susann Ludewig
- Zoological Institute, Division Cellular Neurobiology, TU Braunschweig, Germany
| | - Wenqiang Fan
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Constance Vennin
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany.,German Resilience Center (DRZ), Mainz
| | - Sebastian Loch
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Benedikt Berninger
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany.,Institute of Psychiatry, Psychology & Neuroscience, Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, King's College London, UK
| | - Clementine Hofmann
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - Martin Korte
- Zoological Institute, Division Cellular Neurobiology, TU Braunschweig, Germany.,Helmholtz Centre for Infection Research, Research group Neuroinflammation and Neurodegeneration, Braunschweig, Germany
| | - Ivan Soltesz
- Department of Neurosurgery, Stanford University, USA
| | - Beat Lutz
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany.,German Resilience Center (DRZ), Mainz
| | - Julia Leschik
- Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Germany
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30
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Rosa JM, Pazini FL, Olescowicz G, Camargo A, Moretti M, Gil-Mohapel J, Rodrigues ALS. Prophylactic effect of physical exercise on Aβ 1-40-induced depressive-like behavior: Role of BDNF, mTOR signaling, cell proliferation and survival in the hippocampus. Prog Neuropsychopharmacol Biol Psychiatry 2019; 94:109646. [PMID: 31078612 DOI: 10.1016/j.pnpbp.2019.109646] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 05/02/2019] [Accepted: 05/08/2019] [Indexed: 12/17/2022]
Abstract
Alzheimer's disease (AD) is characterized by progressive cognitive impairments as well as non-cognitive symptoms such as depressed mood. Physical exercise has been proposed as a preventive strategy against AD and depression, an effect that may be related, at least partially, to its ability to prevent impairments on cell proliferation and neuronal survival in the hippocampus, a structure implicated in both cognition and affective behavior. Here, we investigated the ability of treadmill exercise (4 weeks) to counteract amyloid β1-40 peptide-induced depressive-like and anxiety-like behavior in mice. Moreover, we addressed the role of the BDNF/mTOR intracellular signaling pathway as well as hippocampal cell proliferation and survival in the effects of physical exercise and/or Aβ1-40. Aβ1-40 administration (400 pmol/mouse, i.c.v.) increased immobility time and reduced the latency to immobility in the forced swim test, a finding indicative of depressive-like behavior. In addition, Aβ1-40 administration also decreased time spent in the center of the open field and increased grooming and defecation, alterations indicative of anxiety-like behavior. These behavioral alterations were accompanied by a reduction in the levels of mature BDNF and mTOR (Ser2448) phosphorylation in the hippocampus. In addition, Aß1-40 administration reduced cell proliferation and survival in the ventral, dorsal and entire dentate gyrus of the hippocampus. Importantly, most of these behavioral, neurochemical and structural impairments induced by Aβ1-40 were not observed in mice subjected to 4 weeks of treadmill exercise. These findings indicate that physical exercise has the potential to prevent the occurrence of early emotional disturbances associated with AD and this appears to be mediated, at least in part, by modulation of hippocampal BDNF and mTOR signaling as well as through promotion of cell proliferation and survival in the hippocampal DG.
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Affiliation(s)
- Julia M Rosa
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil
| | - Francis L Pazini
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil
| | - Gislaine Olescowicz
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil
| | - Anderson Camargo
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil
| | - Morgana Moretti
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil
| | - Joana Gil-Mohapel
- Division of Medical Sciences, University of Victoria, Island Medical Program, Faculty of Medicine, University of British Columbia, Victoria, British Columbia, Canada
| | - Ana Lúcia S Rodrigues
- Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Florianópolis, Santa Catarina 88040-900, Brazil.
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31
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Platel JC, Angelova A, Bugeon S, Wallace J, Ganay T, Chudotvorova I, Deloulme JC, Béclin C, Tiveron MC, Coré N, Murthy VN, Cremer H. Neuronal integration in the adult mouse olfactory bulb is a non-selective addition process. eLife 2019; 8:44830. [PMID: 31294694 PMCID: PMC6634973 DOI: 10.7554/elife.44830] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Accepted: 07/07/2019] [Indexed: 12/25/2022] Open
Abstract
Adult neurogenesis in the olfactory bulb (OB) is considered as a competition in which neurons scramble during a critical selection period for integration and survival. Moreover, newborn neurons are thought to replace pre-existing ones that die. Despite indirect evidence supporting this model, systematic in vivo observations are still scarce. We used two-photon in vivo imaging to study neuronal integration and survival. We show that loss of new neurons in the OB after arrival at terminal positions occurs only at low levels. Moreover, long-term observations showed that no substantial cell death occurred at later stages. Neuronal death was induced by standard doses of thymidine analogs, but disappeared when low doses were used. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur after neurons reached stable positions. Moreover, this suggests that OB neurogenesis does not represent neuronal turnover but lifelong neuronal addition.
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Affiliation(s)
| | | | - Stephane Bugeon
- Aix-Marseille University, CNRS, IBDM, UMR 7288, Marseille, France
| | - Jenelle Wallace
- Department of Molecular & Cellular Biology, Harvard University, Cambridge, United States
| | - Thibault Ganay
- Aix-Marseille University, CNRS, IBDM, UMR 7288, Marseille, France
| | | | | | | | | | - Nathalie Coré
- Aix-Marseille University, CNRS, IBDM, UMR 7288, Marseille, France
| | - Venkatesh N Murthy
- Department of Molecular & Cellular Biology, Harvard University, Cambridge, United States
| | - Harold Cremer
- Aix-Marseille University, CNRS, IBDM, UMR 7288, Marseille, France
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32
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A balanced evaluation of the evidence for adult neurogenesis in humans: implication for neuropsychiatric disorders. Brain Struct Funct 2019; 224:2281-2295. [PMID: 31278571 DOI: 10.1007/s00429-019-01917-6] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2019] [Accepted: 06/25/2019] [Indexed: 12/17/2022]
Abstract
There is a widespread belief that neurogenesis exists in adult human brain, especially in the dentate gyrus, and it is to be maintained and, if possible, augmented with different stimuli including exercise and certain drugs. Here, we examine the evidence for adult human neurogenesis and note important limitations of the methodologies used to study it. A balanced review of the literature and evaluation of the data indicate that adult neurogenesis in human brain is improbable. In fact, in several high-quality recent studies in adult human brain, unlike in adult brains of other species, neurogenesis was not detectable. These findings suggest that the human brain requires a permanent set of neurons to maintain acquired knowledge for decades, which is essential for complex high cognitive functions unique to humans. Thus, stimulation and/or injection of neural stem cells into human brains may not only disrupt brain homeostatic systems, but also disturb normal neuronal circuits. We propose that the focus of research should be the preservation of brain neurons by prevention of damage, not replacement.
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33
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Qiao H, Li Y, Feng C, Duo S, Ji F, Jiao J. Nap1l1 Controls Embryonic Neural Progenitor Cell Proliferation and Differentiation in the Developing Brain. Cell Rep 2019; 22:2279-2293. [PMID: 29490266 DOI: 10.1016/j.celrep.2018.02.019] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Revised: 12/22/2017] [Accepted: 02/05/2018] [Indexed: 01/27/2023] Open
Abstract
The precise function and role of nucleosome assembly protein 1-like 1 (Nap1l1) in brain development are unclear. Here, we find that Nap1l1 knockdown decreases neural progenitor cell (NPC) proliferation and induces premature neuronal differentiation during cortical development. A similar deficiency in embryonic neurogenesis was observed in Nap1l1 knockout (KO) mice, which were generated using the CRISPR-Cas9 system. RNA sequencing (RNA-seq) analysis indicates that Ras-associated domain family member 10 (RassF10) may be the downstream target of Nap1l1. Furthermore, we found that Nap1l1 regulates RassF10 expression by promoting SETD1A-mediated H3K4 trimethylation at the RassF10 promoter. Nap1l1 KO defects may be rescued by RassF10 overexpression, suggesting that Nap1l1 controls NPC differentiation through RassF10. Our findings reveal an essential role for the Nap1l1 histone chaperone in cortical neurogenesis during early embryonic brain development.
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Affiliation(s)
- Huimin Qiao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yanxin Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chao Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Sino-Danish College at University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuguang Duo
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Fen Ji
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Jianwei Jiao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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34
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Gluncic V, Moric M, Chu Y, Hanko V, Li J, Lukić IK, Lukić A, Edassery SL, Kroin JS, Persons AL, Perry P, Kelly L, Shiveley TJ, Nice K, Napier CT, Kordower JH, Tuman KJ. In utero Exposure to Anesthetics Alters Neuronal Migration Pattern in Developing Cerebral Cortex and Causes Postnatal Behavioral Deficits in Rats. Cereb Cortex 2019; 29:5285-5301. [DOI: 10.1093/cercor/bhz065] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Abstract
During fetal development, cerebral cortical neurons are generated in the proliferative zone along the ventricles and then migrate to their final positions. To examine the impact of in utero exposure to anesthetics on neuronal migration, we injected pregnant rats with bromodeoxyuridine to label fetal neurons generated at embryonic Day (E) 17 and then randomized these rats to 9 different groups receiving 3 different means of anesthesia (oxygen/control, propofol, isoflurane) for 3 exposure durations (20, 50, 120 min). Histological analysis of brains from 54 pups revealed that significant number of neurons in anesthetized animals failed to acquire their correct cortical position and remained dispersed within inappropriate cortical layers and/or adjacent white matter. Behavioral testing of 86 littermates pointed to abnormalities that correspond to the aberrations in the brain areas that are specifically developing during the E17. In the second set of experiments, fetal brains exposed to isoflurane at E16 had diminished expression of the reelin and glutamic acid decarboxylase 67, proteins critical for neuronal migration. Together, these results call for cautious use of anesthetics during the neuronal migration period in pregnancy and more comprehensive investigation of neurodevelopmental consequences for the fetus and possible consequences later in life.
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Affiliation(s)
- V Gluncic
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
- Department of Anesthesiology, Advocate Illinois Masonic Medical Center, Chicago IL, USA
| | - M Moric
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - Y Chu
- Department of Neurological Sciences, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - V Hanko
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
- Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - J Li
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - I K Lukić
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - A Lukić
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - S L Edassery
- Department of Pharmacology, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - J S Kroin
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - A L Persons
- Department of Pharmacology, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
- The Center for Compulsive Behavior and Addiction, Rush University Medical Center, Chicago, IL, USA
| | - P Perry
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - L Kelly
- Department of Neurological Sciences, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - T J Shiveley
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
| | - K Nice
- Department of Neurological Sciences, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - C T Napier
- Department of Pharmacology, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
- The Center for Compulsive Behavior and Addiction, Rush University Medical Center, Chicago, IL, USA
- Department of Psychiatry, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - J H Kordower
- Department of Neurological Sciences, Rush Medical College, Rush University Medical Center, Chicago, IL, USA
| | - K J Tuman
- Department of Anesthesiology, Rush University Medical Center, Chicago, IL, USA
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35
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Carmona-Alcocer V, Rohr KE, Joye DAM, Evans JA. Circuit development in the master clock network of mammals. Eur J Neurosci 2018; 51:82-108. [PMID: 30402923 DOI: 10.1111/ejn.14259] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Revised: 10/08/2018] [Accepted: 10/31/2018] [Indexed: 12/24/2022]
Abstract
Daily rhythms are generated by the circadian timekeeping system, which is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of mammals. Circadian timekeeping is endogenous and does not require exposure to external cues during development. Nevertheless, the circadian system is not fully formed at birth in many mammalian species and it is important to understand how SCN development can affect the function of the circadian system in adulthood. The purpose of the current review is to discuss the ontogeny of cellular and circuit function in the SCN, with a focus on work performed in model rodent species (i.e., mouse, rat, and hamster). Particular emphasis is placed on the spatial and temporal patterns of SCN development that may contribute to the function of the master clock during adulthood. Additional work aimed at decoding the mechanisms that guide circadian development is expected to provide a solid foundation upon which to better understand the sources and factors contributing to aberrant maturation of clock function.
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Affiliation(s)
| | - Kayla E Rohr
- Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin
| | - Deborah A M Joye
- Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin
| | - Jennifer A Evans
- Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin
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LaGamma CT, Tang WW, Morgan AA, McGowan JC, Brachman RA, Denny CA. Antidepressant but Not Prophylactic Ketamine Administration Alters Calretinin and Calbindin Expression in the Ventral Hippocampus. Front Mol Neurosci 2018; 11:404. [PMID: 30459554 PMCID: PMC6232342 DOI: 10.3389/fnmol.2018.00404] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 10/15/2018] [Indexed: 01/20/2023] Open
Abstract
Ketamine has been found to have rapid, long-lasting antidepressant effects in treatment-resistant (TR) patients with major depressive disorder (MDD). Recently, we have also shown that ketamine acts as a prophylactic to protect against the development of stress-induced depressive-like behavior in mice, indicating that a preventative treatment against mental illness using ketamine is possible. While there is significant investigation into ketamine’s antidepressant mechanism of action, little is known about ketamine’s underlying prophylactic mechanism. More specifically, whether ketamine’s prophylactic action is molecularly similar to or divergent from its antidepressant action is entirely unknown. Here, we sought to characterize immunohistochemical signatures of cell populations governing ketamine’s antidepressant and prophylactic effects. 129S6/SvEv mice were treated with saline (Sal) or ketamine (K) either before a social defeat (SD) stressor as a prophylactic, or after SD as an antidepressant, then subsequently assessed for depressive-like behavior. Post-fixed brains were processed for doublecortin (DCX), calretinin (CR) and calbindin (CB) expression. The number of DCX+ neurons in the dentate gyrus (DG) of the hippocampus (HPC) was not affected by prophylactic or antidepressant ketamine treatment, while the number of CR+ neurons in the ventral hilus increased with antidepressant ketamine under SD conditions. Moreover, antidepressant, but not prophylactic ketamine administration significantly altered CR and CB expression in the ventral HPC (vHPC). These data show that while antidepressant ketamine treatment mediates some of its effects via adult hippocampal markers, prophylactic ketamine administration does not, at least in 129S6/SvEv mice. These data suggest that long-lasting behavioral effects of prophylactic ketamine are independent of hippocampal DCX, CR and CB expression in stress-susceptible mice.
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Affiliation(s)
- Christina T LaGamma
- Division of Integrative Neuroscience, Research Foundation for Mental Hygiene, Inc. (RFMH)/New York State Psychiatric Institute (NYSPI), New York, NY, United States
| | - William W Tang
- Department of Psychiatry, Columbia University, New York, NY, United States
| | - Ashlea A Morgan
- Doctoral Program in Neurobiology and Behavior, Columbia University, New York, NY, United States
| | | | - Rebecca A Brachman
- Department of Psychiatry, Columbia University, New York, NY, United States
| | - Christine A Denny
- Division of Integrative Neuroscience, Research Foundation for Mental Hygiene, Inc. (RFMH)/New York State Psychiatric Institute (NYSPI), New York, NY, United States.,Department of Psychiatry, Columbia University, New York, NY, United States
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Aβ42 Peptide Promotes Proliferation and Gliogenesis in Human Neural Stem Cells. Mol Neurobiol 2018; 56:4023-4036. [PMID: 30259399 DOI: 10.1007/s12035-018-1355-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2018] [Accepted: 09/17/2018] [Indexed: 10/28/2022]
Abstract
Amyloid-β 42 [Aβ1-42 (Aβ42)] is one of the main Aβ peptide isoforms found in amyloid plaques of brains with Alzheimer's disease (AD). Although Aβ42 is associated with neurotoxicity, it might mediate several normal physiological processes during embryonic brain development and in the adult brain. However, due to the controversy that exists in the field, relatively little is known about its physiological function. In the present work, we have analyzed the effects of different concentrations of monomeric Aβ42 on cell death, proliferation, and cell fate specification of human neural stem cells (hNSCs), specifically the hNS1 cell line, undergoing differentiation. Our results demonstrate that at higher concentrations (1 μM), Aβ42 increases apoptotic cell death and DNA damage, indicating that prolonged exposure of hNS1 cells to higher concentrations of Aβ42 is neurotoxic. However, at lower concentrations, Aβ42 significantly promotes cell proliferation and glial cell specification of hNS1 cells by increasing the pool of proliferating glial precursors, without affecting neuronal differentiation, in a concentration-dependent manner. At the molecular level, these effects could be mediated, at least in part, by GSK3β, whose expression is increased by treatment with Aβ42 and whose inhibition prevents the glial specification induced by Aβ42. Since the cellular and molecular effects are known to appear decades before the first clinical symptoms, these types of studies are important in discovering the underlying pathophysiological processes involved in the development of AD. This knowledge could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.
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Meyer A, Gläser A, Bräuer AU, Wree A, Strotmann J, Rolfs A, Witt M. Olfactory Performance as an Indicator for Protective Treatment Effects in an Animal Model of Neurodegeneration. Front Integr Neurosci 2018; 12:35. [PMID: 30154701 PMCID: PMC6102364 DOI: 10.3389/fnint.2018.00035] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 07/26/2018] [Indexed: 12/12/2022] Open
Abstract
Background: Neurodegenerative diseases are often accompanied by olfactory deficits. Here we use a rare neurovisceral lipid storage disorder, Niemann–Pick disease C1 (NPC1), to illustrate disease-specific dynamics of olfactory dysfunction and its reaction upon therapy. Previous findings in a transgenic mouse model (NPC1-/-) showed severe morphological and electrophysiological alterations of the olfactory epithelium (OE) and the olfactory bulb (OB) that ameliorated under therapy with combined 2-hydroxypropyl-ß-cyclodextrin (HPßCD)/allopregnanolone/miglustat or HPßCD alone. Methods: A buried pellet test was conducted to assess olfactory performance. qPCR for olfactory key markers and several olfactory receptors was applied to determine if their expression was changed under treatment conditions. In order to investigate the cell dynamics of the OB, we determined proliferative and apoptotic activities using a bromodeoxyuridine (BrdU) protocol and caspase-3 (cas-3) activity. Further, we performed immunohistochemistry and western blotting for microglia (Iba1), astroglia (GFAP) and tyrosine hydroxylase (TH). Results: The buried pellet test revealed a significant olfactory deterioration in NPC1-/- mice, which reverted to normal levels after treatment. At the OE level, mRNA for olfactory markers showed no changes; the mRNA level of classical olfactory receptor (ORs) was unaltered, that of unique ORs was reduced. In the OB of untreated NPC1-/- mice, BrdU and cas-3 data showed increased proliferation and apoptotic activity, respectively. At the protein level, Iba1 and GFAP in the OB indicated increased microgliosis and astrogliosis, which was prevented by treatment. Conclusion: Due to the unique plasticity especially of peripheral olfactory components the results show a successful treatment in NPC1 condition with respect to normalization of olfaction. Unchanged mRNA levels for olfactory marker protein and distinct olfactory receptors indicate no effects in the OE in NPC1-/- mice. Olfactory deficits are thus likely due to central deficits at the level of the OB. Further studies are needed to examine if olfactory performance can also be changed at a later onset and interrupted treatment of the disease. Taken together, our results demonstrate that olfactory testing in patients with NPC1 may be successfully used as a biomarker during the monitoring of the treatment.
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Affiliation(s)
- Anja Meyer
- Institute of Anatomy, Rostock University Medical Center, Rostock, Germany
| | - Anne Gläser
- Institute of Anatomy, Rostock University Medical Center, Rostock, Germany.,Research Group Anatomy, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Anja U Bräuer
- Institute of Anatomy, Rostock University Medical Center, Rostock, Germany.,Research Group Anatomy, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany.,Research Center for Neurosensory Science, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Andreas Wree
- Institute of Anatomy, Rostock University Medical Center, Rostock, Germany
| | - Jörg Strotmann
- Institute of Physiology, University of Hohenheim, Stuttgart, Germany
| | - Arndt Rolfs
- Albrecht-Kossel-Institute for Neuroregeneration, Rostock University Medical Center, Rostock, Germany
| | - Martin Witt
- Institute of Anatomy, Rostock University Medical Center, Rostock, Germany
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Ediriweera MK, Tennekoon KH, Samarakoon SR. In vitro assays and techniques utilized in anticancer drug discovery. J Appl Toxicol 2018; 39:38-71. [DOI: 10.1002/jat.3658] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Revised: 06/01/2018] [Accepted: 06/04/2018] [Indexed: 12/12/2022]
Affiliation(s)
- Meran Keshawa Ediriweera
- Institute of Biochemistry, Molecular Biology and Biotechnology; University of Colombo; Colombo 03 Sri Lanka
| | - Kamani Hemamala Tennekoon
- Institute of Biochemistry, Molecular Biology and Biotechnology; University of Colombo; Colombo 03 Sri Lanka
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40
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Lectins as mitosis stimulating factors: Briefly reviewed. Life Sci 2018; 207:152-157. [DOI: 10.1016/j.lfs.2018.06.003] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Revised: 06/01/2018] [Accepted: 06/04/2018] [Indexed: 01/10/2023]
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Ghosh S, Mandal S, Mandal L. Detecting proliferation of adult hemocytes in Drosophila by BrdU incorporation. Wellcome Open Res 2018; 3:47. [PMID: 29946570 PMCID: PMC5989151 DOI: 10.12688/wellcomeopenres.14560.2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/17/2018] [Indexed: 01/25/2023] Open
Abstract
Drosophila and mammalian hematopoiesis share several similarities that ranges from phases to the battery of transcription factors and signaling molecules that execute this process. These resounding similarities along with the rich genetic tools available in fruitfly makes it a popular invertebrate model to study blood cell development both during normal and aberrant conditions. The larval system is the most extensively studied to date. Several studies have shown that these hemocytes just like mammalian counterpart proliferate and get routinely regenerated upon infection. However, employing the same protocol it was concluded that blood cell proliferation although abundant in larval stages is absent in adult fruitfly. The current protocol describes the strategies that can be employed to document the hemocyte proliferation in adulthood. The fact that a subset of blood cells tucked away in the hematopoietic hub are not locked in senescence, rather they still harbour the proliferative capacity to tide over challenges was successfully demonstrated by this method. Although we have adopted bacterial infection as a bait to evoke this proliferative capacity of the hemocytes, we envision that it can also efficiently characterize the proliferative responses of hemocytes in tumorigenic conditions as well as scenarios of environmental and metabolic stresses during adulthood.
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Affiliation(s)
- Saikat Ghosh
- Developmental Genetics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research-Mohali, Manauli, Punjab, 140306, India
| | - Sudip Mandal
- Molecular Cell and Developmental Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research-Mohali, Manauli, Punjab, 140306, India
| | - Lolitika Mandal
- Developmental Genetics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research-Mohali, Manauli, Punjab, 140306, India
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Lei X, Jiao J. UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling. Stem Cell Reports 2018; 10:1193-1207. [PMID: 29551674 PMCID: PMC5998300 DOI: 10.1016/j.stemcr.2018.02.008] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Revised: 02/11/2018] [Accepted: 02/12/2018] [Indexed: 02/07/2023] Open
Abstract
Neural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.
UTX is essential for cortical development and neuron production Utx suppression impairs normal neocortical neurogenesis in a sex-specific manner UTX affects the levels of H3K27 trimethylation at Pten promoters
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Affiliation(s)
- Xuepei Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jianwei Jiao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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43
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Comparative regenerative mechanisms across different mammalian tissues. NPJ Regen Med 2018; 3:6. [PMID: 29507774 PMCID: PMC5824955 DOI: 10.1038/s41536-018-0044-5] [Citation(s) in RCA: 145] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2017] [Revised: 01/18/2018] [Accepted: 01/23/2018] [Indexed: 02/08/2023] Open
Abstract
Stimulating regeneration of complex tissues and organs after injury to effect complete structural and functional repair, is an attractive therapeutic option that would revolutionize clinical medicine. Compared to many metazoan phyla that show extraordinary regenerative capacity, which in some instances persists throughout life, regeneration in mammalians, particularly humans, is limited or absent. Here we consider recent insights in the elucidation of molecular mechanisms of regeneration that have come from studies of tissue homeostasis and injury repair in mammalian tissues that span the spectrum from little or no self-renewal, to those showing active cell turnover throughout life. These studies highlight the diversity of factors that constrain regeneration, including immune responses, extracellular matrix composition, age, injury type, physiological adaptation, and angiogenic and neurogenic capacity. Despite these constraints, much progress has been made in elucidating key molecular mechanisms that may provide therapeutic targets for the development of future regenerative therapies, as well as previously unidentified developmental paradigms and windows-of-opportunity for improved regenerative repair.
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44
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Hendrickson ML, Zutshi I, Wield A, Kalil RE. Nestin expression and in vivo proliferative potential of tanycytes and ependymal cells lining the walls of the third ventricle in the adult rat brain. Eur J Neurosci 2018; 47:284-293. [PMID: 29359828 DOI: 10.1111/ejn.13834] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Revised: 12/10/2017] [Accepted: 12/19/2017] [Indexed: 12/13/2022]
Abstract
There is a disagreement in the literature concerning the degree of proliferation of cells in the walls of the third ventricle (3rdV) under normal conditions in the adult mammalian brain. To address this issue, we mapped the cells expressing the neural stem/progenitor cell marker nestin along the entire rostrocaudal extent of the 3rdV in adult male rats and observed a complex distribution. Abundant nestin was present in tanycyte cell bodies and processes and also was observed in patches of ependymal cells as well as in isolated ependymal cells throughout the walls of the 3rdV. However, we observed very limited ependymal cell or tanycyte proliferation in normal adult rats as determined by bromodeoxyuridine (BrdU) incorporation or the expression of Ki-67. Moreover, fewer than 13% of the cells that were BrdU-positive (BrdU+) or Ki-67-positive (Ki-67+) expressed nestin. These observations stand in contrast to those made in the subventricular zone of the lateral ventricle (SVZ) and subgranular zone of the hippocampal formation (SGZ), where cell proliferation measured by BrdU incorporation or Ki-67 expression is observed frequently in cells that also express nestin. Thus, while ependymal cell or tanycyte cell proliferation can be promoted by the addition of mitogens, dietary modifications or other in vivo manipulations, the proliferation of ependymal cells and tanycytes in the walls of the 3rdV is very limited in the normal adult male rat brain.
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Affiliation(s)
- Michael L Hendrickson
- School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI, USA
| | - Ipshita Zutshi
- Graduate Program in Biological Sciences, University of California, San Diego, La Jolla, CA, USA
| | - Alyssa Wield
- Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
| | - Ronald E Kalil
- School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI, USA
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45
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Yan XF, Zhao P, Ma DY, Jiang YL, Luo JJ, Liu L, Wang XL. Salvianolic acid B protects hepatocytes from H 2O 2 injury by stabilizing the lysosomal membrane. World J Gastroenterol 2017; 23:5333-5344. [PMID: 28839433 PMCID: PMC5550782 DOI: 10.3748/wjg.v23.i29.5333] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Revised: 03/27/2017] [Accepted: 05/09/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the capability of salvianolic acid B (Sal B) to protect hepatocytes from hydrogen peroxide (H2O2)/carbon tetrachloride (CCl4)-induced lysosomal membrane permeabilization. METHODS Cell Counting Kit-8 assay was used to measure cell viability. Apoptosis and death were assayed through flow cytometry. BrdU incorporation was used to detect cell proliferation. Serum alanine aminotransferase activity and liver malondialdehyde (MDA) content were measured. Liver histopathological changes were evaluated using hematoxylin-eosin staining. Lysosomal membrane permeability was detected with LysoTracker Green-labeled probes and acridine orange staining. The levels of protein carbonyl content (PCC), cathepsins (Cat)B/D, and lysosome-associated membrane protein 1 (LAMP1) were evaluated through western blotting. Cytosol CatB activity analysis was performed with chemiluminescence detection. The mRNA level of LAMP1 was evaluated through quantitative real-time polymerase chain reaction. RESULTS Results indicated that H2O2 induced cell injury/death. Sal B attenuated H2O2-induced cell apoptosis and death, restored the inhibition of proliferation, decreased the amount of PCC, and stabilized the lysosome membrane by increasing the LAMP1 protein level and antagonizing CatB/D leakage into the cytosol. CCl4 also triggered hepatocyte death. Furthermore, Sal B effectively rescued hepatocytes by increasing LAMP1 expression and by reducing lysosomal enzyme translocation to the cytosol. CONCLUSION Sal B protected mouse embryonic hepatocytes from H2O2/CCl4-induced injury/death by stabilizing the lysosomal membrane.
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46
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Molina V, Rodríguez-Vázquez L, Owen D, Valero O, Martí J. Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols. Histochem Cell Biol 2017; 148:477-488. [PMID: 28681271 DOI: 10.1007/s00418-017-1593-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/01/2017] [Indexed: 10/19/2022]
Abstract
An important step in bromodeoxyuridine (BrdU) immunohistochemistry is the production of single-stranded DNA to make the incorporated BrdU accessible to the antibodies. This paper examines the effect of distinct DNA denaturation pretreatments (DNase I, sodium citrate buffer, endonuclease Eco RI and exonuclease III, and HCl hydrolysis) on detection of BrdU. We found that all the methods used in the partial denaturation of DNA combined good nuclear immunostaining with acceptable tissue integrity. We also observed that these immunohistochemical protocols revealed a spatial pattern in the distribution of DNA-synthesizing cells within the cerebellar external granular layer (EGL) of 10-day-old rats, allowing us to estimate the fraction of S-phase cells. Our results indicate that detection of BrdU-stained cells is affected by the distinct histological procedures used in such detection. Additionally, as the duration and phases of the cell cycle in EGL neuroblasts are estimated in accordance with BrdU detection, an effect on this detection can render the measurement of cell cycle inaccurate. The present work shows that DNase I and citrate buffer, at appropriate conditions, may be good alternatives for acid denaturation. However, they are less sensitive than autoradiographic techniques that use 3H-thymidine administration. Finally, current data reveal that short survival times after a single BrdU exposure do not seem to affect the cell cycle progression of the EGL neuroblasts.
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Affiliation(s)
- Vanesa Molina
- Unidad de Citología e Histología, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain
| | - Lucía Rodríguez-Vázquez
- Unidad de Citología e Histología, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain
| | - David Owen
- Departament de Filologia Anglesa i de Germanística, Àrea de Filologia Anglesa, Bellaterra, 08193, Barcelona, Spain
| | - Oliver Valero
- Servei d'Estadística Aplicada, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain
| | - Joaquín Martí
- Unidad de Citología e Histología, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, 08193, Barcelona, Spain.
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47
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Shah D, Ali M, Shukla D, Jain S, Aakalu VK. Effects of histatin-1 peptide on human corneal epithelial cells. PLoS One 2017; 12:e0178030. [PMID: 28542418 PMCID: PMC5441629 DOI: 10.1371/journal.pone.0178030] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2016] [Accepted: 05/07/2017] [Indexed: 11/19/2022] Open
Abstract
Purpose Ocular surface and corneal epithelial wounds are common and potentially debilitating problems. Ideal treatments for these injuries would promote epithelial healing without inflammation, infection and scarring. In addition the best treatments would be cost-efficient, effective, non-toxic and easily applied. Histatin-1 peptides have been shown to be safe and effective enhancers of epithelial wound healing in other model systems. We sought to determine whether histatin-1 peptides could enhance human corneal epithelial wound healing in vitro. Methods Histatin-1 peptides were applied to human corneal epithelial cells and compared over useful dose ranges in scratch assays using time-lapse microscopy. In addition, path finding analysis, cell spreading assays, toxicity and proliferation assays were performed to further characterize the effects of histatin-1 peptide on human corneal limbal epithelial (HCLE). Results Histatin-1 enhanced human corneal epithelial wound healing in typical wound healing models. There was minimal toxicity and no significant enhancement of proliferation of corneal epithelium in response to histatin-1 application. Corneal epithelial spreading and pathfinding appeared to be enhanced by the application of histatin-1 peptides. Conclusions Histatin -1 peptide may enhance migration of HCLE cells and wound healing in vitro. These peptides may have benefit in corneal epithelial wounds and need to be investigated further.
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Affiliation(s)
- Dhara Shah
- Lacrimal Cell Biology Laboratory, University of Illinois at Chicago, Department of Ophthalmology and Visual Sciences, Chicago, United States of America
| | - Marwan Ali
- Lacrimal Cell Biology Laboratory, University of Illinois at Chicago, Department of Ophthalmology and Visual Sciences, Chicago, United States of America
| | - Deepak Shukla
- University of Illinois at Chicago, Department of Ophthalmology and Visual Sciences, Chicago, United States of America
| | - Sandeep Jain
- University of Illinois at Chicago, Department of Ophthalmology and Visual Sciences, Chicago, United States of America
| | - Vinay Kumar Aakalu
- Lacrimal Cell Biology Laboratory, University of Illinois at Chicago, Department of Ophthalmology and Visual Sciences, Chicago, United States of America
- * E-mail:
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Li Y, Jiao J. Histone chaperone HIRA regulates neural progenitor cell proliferation and neurogenesis via β-catenin. J Cell Biol 2017; 216:1975-1992. [PMID: 28515277 PMCID: PMC5496612 DOI: 10.1083/jcb.201610014] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Revised: 01/31/2017] [Accepted: 04/19/2017] [Indexed: 12/14/2022] Open
Abstract
Histone cell cycle regulator (HIRA) is a histone chaperone and has been identified as an epigenetic regulator. Subsequent studies have provided evidence that HIRA plays key roles in embryonic development, but its function during early neurogenesis remains unknown. Here, we demonstrate that HIRA is enriched in neural progenitor cells, and HIRA knockdown reduces neural progenitor cell proliferation, increases terminal mitosis and cell cycle exit, and ultimately results in premature neuronal differentiation. Additionally, we demonstrate that HIRA enhances β-catenin expression by recruiting H3K4 trimethyltransferase Setd1A, which increases H3K4me3 levels and heightens the promoter activity of β-catenin. Significantly, overexpression of HIRA, HIRA N-terminal domain, or β-catenin can override neurogenesis abnormities caused by HIRA defects. Collectively, these data implicate that HIRA, cooperating with Setd1A, modulates β-catenin expression and then regulates neurogenesis. This finding represents a novel epigenetic mechanism underlying the histone code and has profound and lasting implications for diseases and neurobiology.
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Affiliation(s)
- Yanxin Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jianwei Jiao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China .,University of Chinese Academy of Sciences, Beijing 100049, China
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Karasartova D, Gazi U, Tosun O, Gureser AS, Sahiner IT, Dolapci M, Ozkan AT. Anti-Pneumococcal Vaccine-Induced Cellular Immune Responses in Post-Traumatic Splenectomized Individuals. J Clin Immunol 2017; 37:388-396. [PMID: 28488145 DOI: 10.1007/s10875-017-0397-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 04/24/2017] [Indexed: 11/25/2022]
Abstract
PURPOSE Splenectomy is associated with increased risk of overwhelming post-splenectomy infections despite proper anti-pneumococcal vaccination. As most studies concentrated on vaccination-induced humoral immunity, the cellular immune responses triggered in splenectomized patients are not yet well studied. The present study aims to investigate this area as it can contribute to the development of more effective vaccination strategies. METHODS Five healthy and 14 splenectomized patients were vaccinated with pneumococcal conjugate polysaccharide vaccine (PCV) followed by pneumococcal polysaccharide vaccine according to the guidelines established by Advisory Committee on Immunization Practices. PBMC samples collected 0, 8, and 12 weeks after PCV immunization were in vitro stimulated with PCV. Levels of lymphoproliferation, TH cell differentiation, and cytokine release were assessed by carboxyfluorescein succinimidyl ester labeling, intracellular cytokine staining, and ELISA, respectively. RESULTS While TH1-dominated immune response was detected in both groups, asplenic individuals generated significantly lower levels of TH1 cells following in vitro stimulation. Similarly, levels of IFN-γ, IL-4, and IL-17 release and lymphoproliferation were significantly lower in asplenic patients. CONCLUSIONS According to our data, splenectomy negatively influences the levels of PCV-induced lymphoproliferation, TH1 differentiation, and cytokine release. Besides, PCV failed to induce TH17-dominant immune response which is crucial for protection against extracellular pathogens.
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Affiliation(s)
- Djursun Karasartova
- Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum, Turkey
| | - Umut Gazi
- Department of Medical Microbiology and Clinic Microbiology, Faculty of Medicine, Near East University, Near East Boulevard, Nicosia, Cyprus.
| | - Ozgur Tosun
- Department of Biostatistics, Faculty of Medicine, Near East University, Nicosia, Cyprus
| | - Ayse S Gureser
- Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum, Turkey
| | - Ibrahim T Sahiner
- Department of General Surgery, Faculty of Medicine, Hitit University, Corum, Turkey
| | - Mete Dolapci
- Department of General Surgery, Faculty of Medicine, Hitit University, Corum, Turkey
| | - Aysegul T Ozkan
- Department of Medical Microbiology, Faculty of Medicine, Hitit University, Corum, Turkey
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André C, Guzman-Quevedo O, Rey C, Rémus-Borel J, Clark S, Castellanos-Jankiewicz A, Ladeveze E, Leste-Lasserre T, Nadjar A, Abrous DN, Laye S, Cota D. Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation. Diabetes 2017; 66:908-919. [PMID: 27903745 DOI: 10.2337/db16-0586] [Citation(s) in RCA: 126] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Accepted: 11/24/2016] [Indexed: 11/13/2022]
Abstract
Cell proliferation and neuroinflammation in the adult hypothalamus may contribute to the pathogenesis of obesity. We tested whether the intertwining of these two processes plays a role in the metabolic changes caused by 3 weeks of a high-saturated fat diet (HFD) consumption. Compared with chow-fed mice, HFD-fed mice had a rapid increase in body weight and fat mass and specifically showed an increased number of microglia in the arcuate nucleus (ARC) of the hypothalamus. Microglia expansion required the adequate presence of fats and carbohydrates in the diet because feeding mice a very high-fat, very low-carbohydrate diet did not affect cell proliferation. Blocking HFD-induced cell proliferation by central delivery of the antimitotic drug arabinofuranosyl cytidine (AraC) blunted food intake, body weight gain, and adiposity. AraC treatment completely prevented the increase in number of activated microglia in the ARC, the expression of the proinflammatory cytokine tumor necrosis factor-α in microglia, and the recruitment of the nuclear factor-κB pathway while restoring hypothalamic leptin sensitivity. Central blockade of cell proliferation also normalized circulating levels of the cytokines leptin and interleukin 1β and decreased peritoneal proinflammatory CD86 immunoreactive macrophage number. These findings suggest that inhibition of diet-dependent microglia expansion hinders body weight gain while preventing central and peripheral inflammatory responses due to caloric overload.
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Affiliation(s)
- Caroline André
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Omar Guzman-Quevedo
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- Facultad de Químico-Farmacobiología, Universidad Michoacána de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico
| | - Charlotte Rey
- INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
- University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
| | - Julie Rémus-Borel
- INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
- University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
| | - Samantha Clark
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Ashley Castellanos-Jankiewicz
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Elodie Ladeveze
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Thierry Leste-Lasserre
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Agnes Nadjar
- INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
- University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
| | - Djoher Nora Abrous
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
| | - Sophie Laye
- INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
- University of Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France
| | - Daniela Cota
- INSERM, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
- University of Bordeaux, Neurocentre Magendie, Physiopathologie de la Plasticité Neuronale, U1215, Bordeaux, France
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