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      Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases

      review-article
      1 , 2 , 3 , 4 , 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 1 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 15 , 16 , 2 , 5 , 2 , 5 , 33 , 34 , 35 , 36 , 37 , 38 , 34 , 39 , 40 , 1 , 1 , 1 , 34 , 39 , 40 , 2 , 5 , 1 ,
      Cell Death and Differentiation
      Nature Publishing Group UK
      Biochemistry, Neuroscience

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          Abstract

          Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

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          Most cited references147

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          A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.

          Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.
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            Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

            A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.
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              The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1.

              Neurons are known to have a lower glycolytic rate than astrocytes and when stressed they are unable to upregulate glycolysis because of low Pfkfb3 (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3) activity. This enzyme generates fructose-2,6-bisphosphate (F2,6P(2)), the most potent activator of 6-phosphofructo-1-kinase (Pfk1; ref. 4), a master regulator of glycolysis. Here, we show that Pfkfb3 is absent from neurons in the brain cortex and that Pfkfb3 in neurons is constantly subject to proteasomal degradation by the action of the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1. By contrast, astrocytes have low APC/C-Cdh1 activity and therefore Pfkfb3 is present in these cells. Upregulation of Pfkfb3 by either inhibition of Cdh1 or overexpression of Pfkfb3 in neurons resulted in the activation of glycolysis. This, however, was accompanied by a marked decrease in the oxidation of glucose through the pentose phosphate pathway (a metabolic route involved in the regeneration of reduced glutathione) resulting in oxidative stress and apoptotic death. Thus, by actively downregulating glycolysis by APC/C-Cdh1, neurons use glucose to maintain their antioxidant status at the expense of its utilization for bioenergetic purposes.
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                Author and article information

                Contributors
                +353-1-402-2255 , prehn@rcsi.ie
                Journal
                Cell Death Differ
                Cell Death Differ
                Cell Death and Differentiation
                Nature Publishing Group UK (London )
                1350-9047
                1476-5403
                11 December 2017
                March 2018
                : 25
                : 3
                : 542-572
                Affiliations
                [1 ]ISNI 0000 0004 0488 7120, GRID grid.4912.e, Department of Physiology & Medical Physics, , Royal College of Surgeons in Ireland, ; Dublin, Ireland
                [2 ]ISNI 0000 0004 1757 3470, GRID grid.5608.b, Department of Biomedical Sciences, , University of Padova, ; Padova, Italy
                [3 ]ISNI 0000 0001 0942 9821, GRID grid.11804.3c, Department of Medical Biochemistry, , Semmelweis University, ; Budapest, Hungary
                [4 ]ISNI 0000 0000 8954 1233, GRID grid.279863.1, Neuroscience Center of Excellence, , School of Medicine, Louisiana State University Health New Orleans, ; New Orleans, LA 70112 USA
                [5 ]ISNI 0000 0001 1940 4177, GRID grid.5326.2, Neuroscience Institute, , National Research Council (CNR), ; Padova, Italy
                [6 ]ISNI 0000 0001 2183 4846, GRID grid.4711.3, University of Salamanca, CIBERFES, , Institute of Functional Biology and Genomics (IBFG), CSIC, ; 37007 Salamanca, Spain
                [7 ]ISNI 0000 0004 1936 9756, GRID grid.10253.35, Institute of Pharmacology and Clinical Pharmacy, , University of Marburg, ; 35043 Marburg, Germany
                [8 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Neuroregeneration and Stem Cell Programs, , Institute for Cell Engineering, Johns Hopkins University School of Medicine, ; Baltimore, MD 21205 USA
                [9 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Department of Physiology, , Johns Hopkins University School of Medicine, ; Baltimore, MD 21205 USA
                [10 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Department of Neurology, , Johns Hopkins University School of Medicine, ; Baltimore, MD 21205 USA
                [11 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Solomon H. Snyder Department of Neuroscience, , Johns Hopkins University School of Medicine, ; Baltimore, MD 21205 USA
                [12 ]Adrienne Helis Malvin Medical Research Foundation, Diana Helis Henry Medical Research Foundation, New Orleans, LA 70130 USA
                [13 ]ISNI 0000 0001 1034 1720, GRID grid.410711.2, Neuroscience Center, , University of North Carolina, ; Chapel Hill, USA
                [14 ]ISNI 0000000121901201, GRID grid.83440.3b, Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, , University College London, ; London, WC1E 6BT UK
                [15 ]ISNI 0000 0001 2175 4264, GRID grid.411024.2, Department of Anesthesiology and Center for Shock, , Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, ; Baltimore, MD 21201 USA
                [16 ]ISNI 0000 0001 2175 4264, GRID grid.411024.2, Program in Neuroscience, , University of Maryland School of Medicine, ; Baltimore, USA
                [17 ]ISNI 0000 0000 9321 9781, GRID grid.411839.6, Unidad de Neuropsicofarmacología Translacional, , Complejo Hospitalario Universitario de Albacete, ; Albacete, Spain
                [18 ]ISNI 0000 0004 1936 7988, GRID grid.4305.2, UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, ; Edinburgh, EH8 9XD UK
                [19 ]ISNI 0000 0001 2171 9311, GRID grid.21107.35, Department of Molecular Microbiology and Immunology, , Johns Hopkins University, Bloomberg School of Public Health, ; Baltimore, MD 21205 USA
                [20 ]ISNI 0000 0001 2169 2489, GRID grid.251313.7, Department of Biology, , University of Mississippi, ; University, MS 38677 USA
                [21 ]ISNI 0000000419368710, GRID grid.47100.32, Department of Internal Medicine, , Section of Endocrinology, Yale University, ; New Haven, CT USA
                [22 ]ISNI 0000 0001 2194 2329, GRID grid.8048.4, Department of Medical Science-Pharmacology, , University of Castilla-La Mancha, ; Albacete, Spain
                [23 ]ISNI 0000000122199231, GRID grid.214007.0, Neuroscience Translational Center and Depts. of Molecular Medicine and Neuroscience, The Scripps Research Institute, ; La Jolla, CA 92037 USA
                [24 ]GRID grid.465257.7, Neurodegenerative Disease Center, , Scintillon Institute, ; San Diego, CA 92121 USA
                [25 ]ISNI 0000 0001 2107 4242, GRID grid.266100.3, Department of Neurosciences, , University of California San Diego, School of Medicine, ; La Jolla, CA 92093 USA
                [26 ]ISNI 000000041936877X, GRID grid.5386.8, Brain and Mind Research Institute, , Weill Cornell Medicine, ; New York, NY 10065 USA
                [27 ]ISNI 0000 0000 9372 4913, GRID grid.419475.a, National Institute on Aging Intramural Research Program, ; Baltimore, MD USA
                [28 ]ISNI 0000 0001 2264 7217, GRID grid.152326.1, Department of Neurology, , Vanderbilt University School of Medicine, ; Nashville, TN USA
                [29 ]GRID grid.410607.4, University Medical Center Mainz, Dept. of Neurology, ; Mainz, Germany
                [30 ]ISNI 0000 0001 2107 4242, GRID grid.266100.3, Department of Pharmacology, , University of California, ; San Diego, CA 92093 USA
                [31 ]ISNI 0000000121885934, GRID grid.5335.0, MRC Mitochondrial Biology Unit, , University of Cambridge, Cambridge Biomedical Campus, ; Cambridge, CB2 0XY UK
                [32 ]ISNI 0000 0000 8687 5377, GRID grid.272799.0, Buck Institute for Research on Aging, ; Novato, CA 94945 USA
                [33 ]ISNI 0000 0001 2183 4846, GRID grid.4711.3, Dpto. de Biología Molecular, Centro de Biología Molecular Severo Ochoa, , Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, ; 28049 Madrid, Spain
                [34 ]ISNI 0000 0001 2182 2255, GRID grid.28046.38, Brain & Mind Research Institute, , University of Ottawa, ; Ontario, K1H 8M5 Canada
                [35 ]ISNI 0000 0001 2297 6811, GRID grid.266102.1, Department of Neurology, , University of California San Francisco and San Francisco Veterans Affairs Medical Center, ; San Francisco, CA 94121 USA
                [36 ]ISNI 0000 0001 2177 6375, GRID grid.412016.0, The University of Kansas Alzheimer’s Disease Center and Depts. of Neurology, , Molecular and Integrative Physiology, and Biochemistry and Molecular Biology, University of Kansas Medical Center, ; Kansas City, KS 66160 USA
                [37 ]ISNI 0000 0000 8800 7493, GRID grid.410513.2, Pfizer, ; Groton, CT 06340 USA
                [38 ]ISNI 0000 0001 0198 0694, GRID grid.263761.7, Laboratory of Cellular and Molecular Neuropharmacology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, , Soochow University, ; Suzhou, Jiangsu 215021 China
                [39 ]ISNI 0000 0004 0438 0426, GRID grid.424247.3, German Center for Neurodegenerative Diseases (DZNE), ; Bonn, Germany
                [40 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Center for Alzheimer Research, Division of Neurogeriatrics, , Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, ; Stockholm, Sweden
                Author information
                http://orcid.org/0000-0002-6005-1307
                http://orcid.org/0000-0001-9187-3736
                http://orcid.org/0000-0002-5121-5015
                http://orcid.org/0000-0002-2915-3970
                http://orcid.org/0000-0003-2548-4294
                http://orcid.org/0000-0002-8774-0057
                http://orcid.org/0000-0002-8705-3628
                http://orcid.org/0000-0002-4490-3784
                Article
                20
                10.1038/s41418-017-0020-4
                5864235
                29229998
                b1b75681-48b8-4004-bcbe-829ad759341c
                © ADMC Associazione Differenziamento e Morte Cellulare 2017

                Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

                History
                : 11 August 2017
                : 6 October 2017
                : 12 October 2017
                Categories
                Review Article
                Custom metadata
                © The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare 2018

                Cell biology
                biochemistry,neuroscience
                Cell biology
                biochemistry, neuroscience

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