MECANISMOS DE REGULACIÓN DE LA GLUCOLISIS EN NEURONAS Y SU FUNCIÓN EN SUPERVIVENCIA CELULAR PATRICIA RODRÍGUEZ RODRÍGUEZ Salamanca, 2013 Directores: Prof. Dr. D. Juan Pedro Bolaños Hernández Profª. Dra. Dª. Ángeles Almeida Parra Juan Pedro Bolaños Hernández, Catedrático de Bioquímica y Biología Molecular de la Universidad de Salamanca, y Ángeles Almeida Parra, Investigadora del Instituto de Investigación Biomédica de Salamanca y Profesora Asociada del Departamento de Bioquímica y Biología Molecular de la Universidad de Salamanca. AUTORIZAN: La presentación de la Tesis Doctoral titulada “Mecanismos de regulación de la glucolisis en neuronas y su función en supervivencia celular”, que ha sido realizada bajo su dirección por la Licenciada en Farmacia y Bioquímica Dña. Patricia Rodríguez Rodríguez, en el Departamento de Bioquímica y Biología Molecular y en el Instituto de Biología Funcional y Genómica, de la Universidad de Salamanca. En nuestra opinión, reúne todos los requisitos científicos y formales para ser defendida y optar al Título de Doctor Europaeus. Salamanca, a 17 de Junio de 2013 Fdo.: Juan Pedro Bolaños Hernández Fdo.: Ángeles Almeida Parra INDEX INDEX 1. INTRODUCTION .......................................................................................................1 1. GLUCOSE METABOLISM IN THE CENTRAL NERVOUS SYSTEM ..................................... 3 1.1. GLUCOSE TRANSPORTERS ......................................................................................................... 4 1.2. GLUCOSE METABOLIC PATHWAYS ............................................................................................. 5 1.3. LACTATE CONSUMPTION IN NEURONS AND THE ASTROCYTE-NEURON LACTATE SHUTTLE HYPOTHESIS. ..................................................................................................................... 15 1.4. REACTIVE OXYGEN SPECIES (ROS) GENERATION AND DETOXIFICATION ......................... 17 2. GLUTAMATERGIC NEUROTRANSMISSION ...................................................................... 20 2.1. GLUTAMATE ................................................................................................................................. 20 2.2. GLUTAMATE RECEPTORS .......................................................................................................... 20 2.3. GLUTAMATE TRANSPORTERS ................................................................................................... 23 2.4. GLUCOSE METABOLISM IN GLUTAMATERGIC NEUROTRANSMISSION ................................ 24 2.5. EXCITOTOXICITY ......................................................................................................................... 26 2. HYPOTHESIS AND OBJECTIVES .........................................................................31 3. MATERIALS AND METHODS ................................................................................35 1. PLASMID CONSTRUCTIONS, AMPLIFICATION AND PURIFICATION ............................. 37 1.1. pEGFP-C1-TIGAR PLASMID CONSTRUCTION ........................................................................... 37 1.2. G6PD, PFKFB3 and mutPFKFB3 PLASMID CONSTRUCTIONS .................................................. 38 1.3. BACTERIAL TRANSFORMATION AND PLASMIDS PURIFICATION ........................................... 39 2. “SMALL INTERFERING RNA” (SIRNA) DESIGN ................................................................ 39 3. ANIMALS. .............................................................................................................................. 40 4. CELL CULTURE .................................................................................................................... 41 4.1. CORTICAL NEURONS IN PRIMARY CULTURE........................................................................... 41 4.2. ASTROCYTES IN PRIMARY CULTURE ....................................................................................... 42 4.3. HEK-293T ...................................................................................................................................... 42 5. CELL TREATMENTS ............................................................................................................. 43 5.1 CELL TRANSFECTIONS ................................................................................................................ 43 5.2 NMDA RECEPTORS STIMULATION ............................................................................................. 43 5.3 INHIBITION OF PPP ACTIVITY AND MITOCHONDRIAL PYRUVATE UPTAKE. .......................... 44 6. DETERMINATION OF Ca2+ UPTAKE ................................................................................... 44 7. ELECTROPHORESIS AND PROTEIN IMMUNODETECTION (WESTERN BLOT) ............ 45 INDEX 8. PROTEIN IMMUNOPRECIPITATION. .................................................................................. 46 9. REVERSE TRANSCRIPTION-PCR (RT-PCR). .................................................................... 47 10. FLOW CYTOMETRIC ANALYSIS OF APOPTOTIC CELL DEATH .................................. 48 11. DETECTION OF REACTIVE OXYGEN SPECIES (ROS) ................................................... 49 12. DETERMINATION OF METABOLITES .............................................................................. 49 12.1. D-GLUCOSE ................................................................................................................................ 49 12.2. L-LACTATE .................................................................................................................................. 50 12.3. GLUCOSE-6-PHOSPHATE (G6P) ............................................................................................... 50 12.4. GLUTATHIONE ............................................................................................................................ 51 12.5. FRUCTOSE-2,6-BISPHOSPHATE............................................................................................... 52 13. PGI ACTIVITY DETERMINATION ...................................................................................... 53 14. PFK-1 ACTIVITY DETERMINATION .................................................................................. 53 15. GLYCOLYTIC FLUX ASSESMENT .................................................................................... 54 16. PENTOSE-PHOSPHATE PATHWAY (PPP) FLUX MEASUREMENTS ............................ 55 17. IMMUNOCYTOCHEMISTRY ............................................................................................... 59 18. CONFOCAL MICROSCOPY OF TRANSFECTED CELLS. ............................................... 59 19. STATISTICAL ANALYSIS................................................................................................... 59 4. RESULTS ............................................................................................................... 61 1. GLYCOLYTIC FLUX INCREASES BY INHIBITING PENTOSE-PHOSPHATE PATHWAY (PPP) OR MITOCHONDRIAL PYRUVATE UPTAKE IN NEURONS. ...................................... 63 2. THE RATE OF GLUCOSE OXIDIZED THROUGH THE PPP IS INHIBITED BY DHEA AND NOT BY HCN. ............................................................................................................................ 64 3. PHOSPHOGLUCOSE ISOMERASE (PGI) IS A HIGHLY ACTIVE ENZYME IN NEURONS. ................................................................................................................................................... 66 4. KNOCK-DOWN OF PGI INCREASES PPP ACTIVITY ........................................................ 67 5. EFFECT OF DHEA AND HCN ON GLUCOSE-6-PHOSPHATE CONCENTRATION ......... 69 6. EFFECT OF DHEA AND HCN ON EXTRACELLULAR AND INTRACELLULAR LACTATE CONCENTRATIONS ................................................................................................................. 69 7. CORTICAL PRIMARY NEURONS RESPOND TO GLUTAMATE RECEPTORS ACTIVATION BY INCREASING INTRACELLULAR Ca2+ LEVELS. ....................................... 71 8. NMDAR STIMULATION PROMOTES PROTEIN STABILIZATION OF THE GLYCOLYTIC- PROMOTING ENZYME PFKFB3 IN NEURONS. ..................................................................... 73 INDEX 9. NMDAR STIMULATION DOES NOT ALTER THE PFKFB3 mRNA LEVELS IN NEURONS. .................................................................................................................................................... 74 10. NMDAR STIMULATION TRIGGERS NUCLEUS-TO-CYTOSOL PFKFB3 TRANSLOCATION..................................................................................................................... 75 11. NMDAR STIMULATION INCREASES THE RATE OF GLYCOLYSIS AND DECREASES THE RATE OF PPP THROUGH PFKFB3. ................................................................................ 78 12. NMDAR STIMULATION LEADS TO IMPAIRMENT OF GLUTATHIONE REGENERATION THAT IS MEDIATED BY PFKFB3 STABILIZATION. ............................................................... 80 13. THE PPP TO GLYCOLYSIS SHIFT CAUSED BY NMDAR STIMULATION TRIGGERS OXIDATIVE STRESS ................................................................................................................. 81 14. NMDAR ACTIVATION TRIGGERS APOPTOTIC DEATH BY SWITCHING PPP TO GLYCOLYSIS. ............................................................................................................................ 83 15. EXPRESSION OF A MUTANT FORM OF PFKFB3 INSENSITIVE TO APC/C-Cdh1 MIMICS NMDAR AT CAUSING OXIDATIVE