Cytochrome c oxidase mediates the regulation of mitochondrial function in an in vitro model of Huntington's disease [Elektronische Ressource] / vorgelegt von Shilpee Singh

Sunny Oasis

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Cytochrome c oxidase mediates the regulation of mitochondrial function in an in vitro model of Huntington’s disease Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Shilpee Singh, M.Sc. Biotechnology aus Patna, India Berichter: Universitätsprofessor Dr. Hermann Wagner Universitätsprofessor Dr. Cordian Beyer Tag der mündlichen Prüfung: 08.10.2009 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. i Contents Abbreviations iii 5 1 Introduction 1.1 Brain: The different cell types and their functions 5 1.2 Neurodegeneration and brain region specificity 6 1.3 Huntington’s disease 7 1.3.1 Huntington’s disease - The in vivo model 7 1.3.2 Application of 3-nitropropionic acid (NPA) – The in vitro model 7 1.3.3 Impairment of mitochondrial function in Huntington’s Disease (HD) 8 1.4 The role of mitochondria in neurodegenerative diseases 9 1.5 Cytochrome c oxidase (COX) 10 1.5.1 Enzyme reaction 10 1.5.

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 1 Mo

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  Cytochrome c oxidase mediates the regulation of mitochondrial function in anin vitromodel of Huntingtons disease

  Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen  University zur Erlangung des akademischen Grades eines Doktors der  Naturwissenschaften genehmigte Dissertation   vorgelegt von  Shilpee Singh, M.Sc. Biotechnology  aus Patna, India   Berichter: Universitätsprofessor Dr. Hermann Wagner  Universitätsprofessor Dr. Cordian Beyer   Tag der mündlichen Prüfung: 08.10.2009      Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online  rgüabevfr. 

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 Contents   Abbreviations   1 Introduction 1.1 Brain: The different cell types and their functions 1.2 Neurodegeneration and brain region specificity 1.3 Huntingtons disease 1.3.1 Huntington’s disease - The in vivo model 1.3.2 Application of 3-nitropropionic acid (NPA) – The in vitro model 1.3.3 Impairment of mitochondrial function in Huntington’s Disease (HD) 1.4 The role of mitochondria in neurodegenerative diseases 1.5 Cytochrome c oxidase (COX) 1.5.1 Enzyme reaction 1.5.2 Enzyme subunit IV and its isoforms 1.6 Aims of the thesis  2 Materials and methods 2.1 Cell culture 2.1.1 Preparation of primary astrocytic culture 2.1.2 Preparation of primary neuronal cultures 2.1.3 Transfection and small interference (siRNA)-mediated knock-down 2.2 Cell treatment and staining 2.2.1 Immunocytochemistry 2.2.2 Hoechst and propidium iodide staining 2.2.3 Trypan blue staining for astrocytic culture 2.2.4 Trypan blue staining for neurons 2.3 Molecular biology 2.3.1 RNA isolation 2.3.2 Reverse transcription 2.3.3 Quantitative real time-PCR analysis 2.4 Colorimetric, fluorometric and luminometric assays 2.4.1 Intracellular ATP determination 2.4.2 Hydrogen peroxide detection in mitochondria 2.4.3 Measurement of reactive oxygen species (ROS) 2.4.4 Glycogen assay 2.4.5 Protein determination 2.5 Mitochondria isolation and enzyme kinetics measurement 2.5.1 Preparation of mitochondria from primary astrocytes 2.5.2 Polarographic measurements 2.6 Statistical analysis  3. Cytochrome c oxidase isoform IV-2 is involved in 3-nitropropionic  acid-induced toxicity in striatal astrocytes 3.1 Abstract 3.2 Introduction 3.3 Methods and material 3.4 Results 3.5 Discussion

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iii  5 5 6 7 7 7 8 9 10 10 11 13  14 14 14 14 16 16 16 18 19 19 19 19 20 20 21 21 22 22 23 23 23 23 24 24  25 25 25 26 29 38

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 4. Brain region specificity of 3-nitropropionic acid-induced  vulnerability of neurons involves cytochrome c oxidase 4.1 Abstract 4.2 Introduction 4.3 Methods and material 4.4 Results 4.5 Discussion  General discussion  Summary and conclusions  Outlook and future perspective  References  Zusammenfassung und Schlußfolgerung     Acknowledgement   Curriculum vitae                                                  

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42 42 42 44 46 51  54  57  58  59  70

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    Abbreviations AD  ADP  ATP  BBB  BCA  BSA  cDNA  CNS  COX Ct DEPC DMEM DMSO  DNA  dNTP  DTT  E  EDTA  ETC  FCS  GABA  GAPDH  GFAP  H 2 DCF-DA  HD  HEPES  HPRT  K m  MEM  NBM  NeuN  NPA  PBS 

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Alzheimers disease  Adenosine-di-phosphate  Adenosine-tri-phosphate  Blood brain barrier  Bovine calf serum  Bovine serum albumin  Complementary DNA  Central nervous system  Cytochrome c oxidase threshold cycle Diethyl pyrocarbonate Dulbeccos modified Eagles medium Dimethyl sulfoxide  Deoxyribonucleic acid  2-deoxyribonucleoside-5-tri-phosphate  Dithiotreitol  Embryonal day  Ethylenediamine tetraacetic acid  Electron transport chain  Fetal calf serum  γ-aminobutyric acid  Glyceraldehyde 3-phosphate dehydrogenase  Glial fibrillary acidic protein  2'7'-dichlorodihydrofluorescein diacetate acetyl ester  Huntingtons disease  N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid  Hypoxanthine guanine phosphoribosyl transferase  Michaelis-Menten constant  Minimum essential medium  Neuronal basal medium  Neuronal N  Nitropropionic acid  Phosphate buffered saline 

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PCR PD PI PLO PMSF ROS rpm RT-PCR SDS siRNA SSC TN Tris-HCl V max  

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Polymerase chain reaction Parkinsons disease Propidium iodide Poly-L-ornithine Phenylmethylsulfonylfluoride Reactive oxygen species rotations per minute Real-time polymerase chain reaction Sodium dodecyl sulphate small-interfering ribonucleic acid saline-sodium citrate Turnover number Tris-(hydroxymethyl) aminomethane-hydrogen chloride Maximal activity

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 1. Introduction  1.1   Brain: The different cell types and their functions The brain is composed of mainly two classes of cells, neurons and glia. Glial cells outnumber neurons by 10:1 (sfn.org; Society for Neuroscience, 2000). So far, studies on brain function have been mainly focused on the effects of toxic and degenerative processes on the survival of neurons because of their crucial involvement in the chemical and electrical transmission of information between cells and over long distances. However, with course of time, glial cells too have emerged as new focus. Glial cells are non-neuronal cells that fulfill a large number of various functions including the formation of the blood-brain-barrier, providing nutrients and growth factors to the nervous tissue, and playing a principal role in repair processes in the brain and spinal cord following traumatic injuries (Davieset al.2006). Astrocytes belong to the class of glial cells and are characterized by a star-shaped morphology. They form close contacts to neighboring cells, like other astrocytes, blood vessel, neurons, and synaptic clefts by enwrapping these structures with their processes. There is a growing body of evidence that astrocytes provide many different and essential functions to support neuronal survival (Jourdainet al. Pellerin 2007;et al. Astrocytes also play an important role in 2007). neurodegenerative diseases. It is well-acknowledged that interactions between neurons and astrocytes occur and are critical for cell-cell signaling, energy metabolism, extracellular ion homeostasis, volume regulation, and neuroprotection in the central nervous system (Bezziet al. 2001; Haydonet al. 2001) For instance, astrocytes were shown to protect neurons against glutamate toxicity (Rosenberg and Aizenman, 1989) and peroxide generation (Deshagaret al. 1996). Astrocytes do express proteins that are necessary for the uptake of glutamate at synapses, ammonia detoxification, buffering of extracellular K + , and volume regulation (Hertzet al. 2004; Newmanet al. 2003). Astrocytes are also responsible for detection, propagation, and modulation of excitatory synaptic signals (Maragakis and Rothstein, 2006). With respect to the high energy demand of neuronal signaling processes, astrocytes provide an essential metabolic support to the neurons, thereby sustaining brain activity.  

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   Fig. 1 Functional diversity of astrocytes Astrocyte functions include the modulation of synaptic (1) function via glutamate transporters, which remove glutamate from the synaptic cleft into the cell. (2) Communication between astrocytes occurs via release and binding of ATP to purine receptors on adjacent astrocytes. ATP binding results in phospholipase C activation with subsequent downstream activation of inositol triphosphate resulting in calcium mobilization. (3) Gap junctions contribute to an astrocyte syncytium for the exchange of small molecules and cellcell-communication. Metabolic functions include (4) the replenishment of neuronal glutamate via the glutamateglutamine-cycle, and (5) the transport of glucose from the vasculature to neural cells. (6) The regulation of blood flow is modulated by astrocyte end-feet enwrapping blood vessels including vasodilation being mediated by the release of vasoactive substances. (7) Glutamate release might occur following elevations in intracellular calcium and the activation of other factors related to prostaglandins. (8) Glutamate release through hemichannels can be inducedin vitrothrough lowering the extracellular calcium concentration. (9) Glutamate binding to metabotropic glutamate receptors activates intracellular calcium leading to the release of vasodilatory substances. Abbreviations: Gln, glutamine; Glu, glutamate; IP3 ,  inositol triphosphate; PLC, phospholipase C (modified from Maragakis and Rothstein, 2006).   1.2   Neurodegeneration and brain region specificity  Neurodegeneration implies an impairment of neuronal function culminating often in a progressive loss of neurons. Many neurodegenerative diseases including Huntingtons, Parkinsons, and Alzheimers disease occur as a result of progressive loss of neurons affecting different brain regions to a different extent. Huntingtons disease causes astrogliosis and a loss of medium spiny neurons in the striatum   (Vonsattelet al.1985; Brouilletet al.1999, Viset al.1999). Neural cells in the striatum are mainly affected, but cells of the frontal and temporal cortices are also involved (Selemonet al.2003). Alzheimer's disease is characterized by a loss of neurons in