Allma Mater Studiiorum – Uniiversiità dii Bollogna DOTTORATO DI RICERCA IN Biologia Cellulare e Molecolare Ciclo XXIX Settore Concorsuale di afferenza: 05/D1 Settore Scientifico disciplinare: BIO/09 FISIOLOGIA TITOLO TESI PHYSIOPATHOLOGICAL PROTEIN RELEASE BY GLIAL CELLS: FOCUS ON PURINE NUCLEOSIDE PHOSPHORYLASE (PNP), SUPEROXIDE DISMUTASE 1 (SOD1), AND α-SYNUCLEIN. Presentata da: Francesca Massenzio Coordinatore Dottorato Relatore Chiar.mo Prof. Giovanni Capranico Prof.ssa Barbara Monti Esame finale anno 2017 INDEX ABSTRACT ..................................................................................................................................................... 4 1. INTRODUCTION ......................................................................................................................................... 8 1.1 THE CENTRAL NERVOUS SYSTEM ........................................................................................................ 8 1.1.1 MICROGLIAL CELLS ............................................................................................................................. 9 1.1.1.1 Origin of microglia ......................................................................................................................................10 1.1.1.2 Microglial phenotypes ................................................................................................................................11 1.1.1.3 Physiological functions of microglia ...........................................................................................................11 1.1.1.4 Pathological functions of microglia ............................................................................................................12 1.1.2 ASTROCYTES ..................................................................................................................................... 16 1.1.2.1 Physiological role of astrocytes ..................................................................................................................17 1.1.2.2 Pathological role of astrocytes ...................................................................................................................19 1.1.3 NEURON-GLIA INTERACTIONS .......................................................................................................... 20 1.1.4 GLIAL CELL SECRETION ..................................................................................................................... 22 1.1.4.1 Glial vescicular release mediated by physiopathologycal stimuli ...............................................................22 1.1.4.2 Vescicule-mediated release of proteins .....................................................................................................24 1.2 PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) ....................................................................................26 1.2.1 PNP DEFICIENCY ............................................................................................................................... 27 1.3 SUPEROXIDE DISMUTASE 1 (SOD1) ....................................................................................................28 1.3.1 AMYOTROPHIC LATERAL SCLEROSIS (ALS) ....................................................................................... 30 1.3.2 MUTANT SOD1 IN ALS ...................................................................................................................... 33 1.3.3 GLIAL CELL INVOLVEMENT IN ALS .................................................................................................... 34 1.4 ALPHA-SYNUCLEIN (Α-SYN) ..................................................................................................................36 1.4.1 PARKINSON’S DISEASE ...................................................................................................................... 38 1.4.2 MUTANT α-syn IN PARKINSON’S DISEASE ........................................................................................ 40 1.4.3 GLIAL CELL INVOLVEMENT IN PARKINSON’S DISEASE ...................................................................... 42 2. AIMS ........................................................................................................................................................44 3. MATERIAL AND METHODS .......................................................................................................................48 3.1 PRIMARY RAT CULTURE OF MICROGLIA AND ASTROCYTES .................................................................48 3.2 PRIMARY RAT CULTURE OF CEREBELLAR GRANULE NEURONS (CGNS) .................................................50 3.3 IMA 2.1 AND N9 IMMORTALIZED CELLS LINES .....................................................................................52 3.4 CULTURE TREATMENTS ......................................................................................................................52 3.4.1 TREATMENT WITH LPS ..................................................................................................................... 52 3.4.2 TREATMENT WITH INTERFERON- γ (IFN- γ) ...................................................................................... 53 1 3.4.3 TREATMENT WITH ADENOSINE TRIPHOSPHATE (ATP) AND 2'-3'-O-(4- BENZOYLBENZOYL)-ATP (BZ-ATP) ............................................................................................................................ 53 3.4.5 L-SERINE TREATMENT ...................................................................................................................... 54 3.4.6 TREATMENT WITH THREALOSE ........................................................................................................ 55 3.5 GLIAL CELL TRANSFECTION .................................................................................................................55 3.5.1 PURIFICATION OF PLASMID DNA ..................................................................................................... 55 3.5.2 TRANSFECTION OF GLIAL PRIMARY CULTURES ................................................................................ 56 3.5.3 TRANSFECTION OF GLIAL CELL LINES ............................................................................................... 57 3.6 CELLS AND CONDITIONED MEDIA .......................................................................................................58 3.7 PURIFICATION OF EXTRACELLULAR VESICLES FROM CONDITIONED MEDIUM .....................................58 3.8 CO- CULTURE OF CEREBELLAR GRANULE NEURONS (CGNS) WITH PRIMARY MICROGLIA OR ASTROCYTES OVEREXPRESSING WT/ MUTSOD1 ......................................................................................................59 3.9 IMMUNOCYTOCHEMISTRY .................................................................................................................59 3.10 NITRIC OXIDE DETECTION ASSAY ......................................................................................................61 3.11 HOECHST STAINING AND NUCLEI COUNTING ....................................................................................61 3.12 WESTERN BLOT ................................................................................................................................62 3.13 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ...............................................................64 3.14 QUANTITATIVE REAL TIME PCR .........................................................................................................65 3.15 STATISTICAL ANALISYS .....................................................................................................................66 4. RESULTS ...................................................................................................................................................67 4.1 PNP RELEASE BY GLIAL CELLS ..............................................................................................................67 4.1.1 PNP EXPRESSION AND RELEASE BY RAT PRIMARY CULTURES OF MICROGLIA, ASTROCYTES AND DIFFERENTIATED CGNS .............................................................................................................. 67 4.1.2 INFLAMMATORY STIMULI DO NOT MODIFY CONSTITUTIVE PNP SECRETION FROM CULTURED MICROGLIA, ASTROCYTES AND DIFFERENTIATED CGNS ....................................................................... 69 4.1.3 ATP ENHANCES CONSTITUTIVE PNP SECRETION FROM CULTURED MICROGLIA AND ASTROCYTES ............................................................................................................................................................ 72 4.1.4 PNP RELEASE IS MEDIATED VIA THE LYSOSOMAL SECRETORY PATHWAY ....................................... 73 4.1.5 INVOLVEMENT OF P2X7 RECEPTORS IN PNP RELEASE ..................................................................... 76 4.2 SOD1 RELEASE FROM RAT PRIMARY CULTURES OF MICROGLIA AND ASTROCYTES IN PHYSIOPATHOLOGICAL CONDITIONS ....................................................................................................................80 4.2.1 MICROGLIAL SOD1 RELEASE THROUGH LYSOSOMES ....................................................................... 80 4.2.2 INTRACELLULAR MUTANT SOD1 ACCUMMULATION IN PRIMARY CULTURES OF MICROGLIA AND ASTROCYTES ................................................................................................................................ 83 4.2.3 AUTOPHAGIC PATHWAY ALTERATION