Discipline: Neurosciences Ecole Doctorale Vie et Santé

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Niveau: Supérieur, Doctorat, Bac+8
       Thesis presented for the degree of  DOCTOR OF PHILOSOPHY EN CO?TUTELLE of      UNIVERSITE LOUIS PASTEUR  FRANCE  Discipline: Neurosciences  Ecole Doctorale Vie et Santé    &    UNIVERSITÄT BASEL  SWITZERLAND  Discipline : Neurosciences  Philosophisch?Naturwissenschaftliche Fakultät        Neuronal death mechanisms in cerebellar  Purkinje cells    by    Stéphane HEITZ      Defended September 22nd 2008 in front of:    Dr. Patrice Codogno  External reporter  Pr. Peter Scheiffele  External reporter  Pr. Pierrick Poisbeau  ULP internal reporter  Pr. Markus Ruegg  UniBasel internal reporter  Dr. Fekrije Selimi  External examiner  Dr. Yannick Bailly  PhD co?supervisor  Pr. Josef Kapfhammer  PhD co?supervisor  Dr. Bernard Poulain  PhD co?supervisor 

  • dr. bernard poulain 

  • recherche en sciences

  • heitz - centre national de la recherche scientifique

  • heitz

  • external examiner  dr. yannick bailly 

  • interventions enflammées sur l'allongement du temps de travail


Publié le : mercredi 20 juin 2012
Lecture(s) : 113
Source : scd-theses.u-strasbg.fr
Nombre de pages : 155
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ThesispresentedforthedegreeofDOCTOROFPHILOSOPHYENCOTUTELLEofUNIVERSITELOUISPASTEURFRANCEDiscipline:NeurosciencesEcoleDoctoraleVieetSanté&UNIVERSITÄTBASELSWITZERLANDDiscipline:NeurosciencesPhilosophischNaturwissenschaftlicheFakultätNeuronaldeathmechanismsincerebellarPurkinjecellsbyStéphaneHEITZnd DefendedSeptember222008infrontof:Dr.PatriceCodognoExternalreporterPr.PeterScheiffeleExternalreporterPr.PierrickPoisbeauULPinternalreporterPr.MarkusRueggUniBaselinternalreporterDr.FekrijeSelimiExternalexaminerDr.YannickBaillyPhDcosupervisorPr.JosefKapfhammerPhDcosupervisorDr.BernardPoulainPhDcosupervisor
Stipend support:
Supervision:
Contributionofothers
Statistical support:
- Neurex Network to S. Heitz -Roche Research Foundation to S. Heitz -Fondation Novartis pour la Recherche en Sciences Biomédicales to S. Heitz
Western Blotting support:
Genotyping support:
Project costs:
Dr Y. Bailly and Prof J. Kapfhammer
Dr J-L Rodeau
Dr N. Grant
Vanessa Gautheron, Drs Yves Lutz and Jean-Paul Fuchs
30000€
Project funds: -G.I.S. Infections à prions to Dr Y. Bailly-Travel Grant Région Alsace to S. Heitz-Travel Grant Sociétédes Neurosciences to S. Heitz-Centre National de la Recherche Scientifique funds to Dr Y. Bailly -University funds, Université Louis Pasteur to S. Heitz-University funds, Universität Basel to Prof J. Kapfhammer and S. Heitz
Infrastructures: -Institut des Neurosciences Cellulaires et Intégratives, Département Neurotransmission et Sécrétion Neuroendocrine, UMR 7168/LC2, CNRS and Université Louis Pasteur -Anatomisches Institut der Universität Basel -Plateforme d’Imageriein vitro, IFR 37 des Neurosciences -Animal facility at the Anatomisches Institut der Universität Basel -Plateforme d’Exploration Fonctionnelle, IFR 37 des Neurosciences
Acknowledgments
The work presented in this thesis was carried out in cotutelle between the Anatomisches Institut der Universität Basel and the Départment Neurosécrétion et Sécrétion Neuroendocrine of INCI in Strasbourg managed by Dr Marie-France Bader. I thank her for warm welcome during my DEA and my thesis. I thank Pr Josef Kapfhammer for his engagement and his participation in the co-direction of my thesis and the enriching discussions for both scientific and technical aspects. I thank Dr Yannick Bailly for encouraging me from the first day of my DEA and the fascinating scientific adventure of the last 4 years and Dr Bernard Poulain for accepting to be my co-supervisor. I wish to thank my jury members Drs Fekrije Selimi and Patrice Codogno, Prs Peter Scheiffele, Pierrick Poisbeau and Markus Ruegg for their interest in my work and for judging. A special thank to Dr Jean Mariani for his implication in my work. Many scientific thanks for advice to Dr Hadi Zanjani for the quantification method of Purkinje cells, to Dr Nancy Grant for the intricacies of a Western blot and how to write a an comprehensible English, Dr Laure Rondi-Reigg for the secrets of footprint tests and Dr Jean-Luc Rodeau for statistical analysis. I wish to thank Brenda Bonnici and Vesna Radojevic for sharing their benches when the female mice delivered at an unexpected moment and Markus Saxer for technical help. A special happy thought to Brenda for those fascinating debates concerning the European Union. Many thanks to Vanessa Gautheron who constantly made genotypes for me and revealed the mystery of the SSCP and to Drs Yves Lutz and Jean-Paul Fuchs for their expertise in Dpl genotyping.  Many thanks to Fabrice Richard and Raphael Leschiera who helped me during their training courses in cutting some mouse brains and counting autophagic profiles in ultrastructural sections, respectively.  I also wish to thank Dr Sophie Reibel-Foisset, Dr Dominique Ciocca and Nicolas Lethenet for their technical help in the animal facility.
Je tiens à remercier mon petit rossignol alsacien, Anne-Marie, qui a supporté mes blagues pendant 5 longues années. Blagues qui n’étaient qu’une réplique à ses trémolos et concerts improvisés de Poulenc ou de chant lyrique scandinaves. Sache que tes essais culinaires me manqueront grandement. Un grand merci à Guy, dont j’ai supporté les blagues pendant 5 ans, pour son immense savoir et pour toute son aide technique. Je n’oublierai pas tes interventions enflammées sur l’allongement du temps de travail. Puisses-tu profiter pleinement et heureusement de ta retraite le jour venu. Egalement mille mercis à Monique et surtout Valérie à qui j’ai décommandé sans relâche des séances de microscopie et qui, avec une constance inébranlable, a fait semblant de croire à mes excuses douteuses. Les pauses café-bredele me laissent un souvenir impérissable. Je remercie grandement tous les membres de NSN et plus particulièrement Jean-Luc Dupont pour nos traits d’humour concernant le « Joe Bar Team », Renaud pour nos discussions doctorales, Fanny, Aurore, Aurélie, Petra, Valérie, Frédéric (x2), Cédric, Alexandre, Yann, Etienne, Stéphane (x2), Nicolas pour nos discussions tout court. Merci au Dr Michael Gutnic pour m’avoir donné l’opportunité d’enseigner les statistiques aux étudiants de cycle de Licence. Merci également à tous ceux avec qui j’ai collaboré au sein de l’Addal et surtout Jennifer pour son indéfectible amitié. Merci surtout à ma maman et à Eleonore, mon épouse, pour m’avoir encouragé durant cette période. Merci à ma petite Camille de m’avoir laissé dormir la nuit, lorsque je rédigeais le présent manuscrit. Je ne saurais finir ces remerciements sans mentionner le grand Albert Einstein dont la théorie de la relativité restreinte à grandement contribué à sauver mes années de lycée ainsi qu’Olivier Combeau, enseignant de biologie au Séminaire de Jeunes de Walbourg, pour avoir cru en moi dès le début et m’avoir poussé dans cette voie alors que je n’étais qu’un petit adolescent insolent.
Tableofcontents
Abbreviations............................................................................................................... 1Introduction ................................................................................................................. 31.Molecularbasisofprogrammedneuronalcelldeath ............................................ 71.1Apoptosis:thetypeIprogrammedcelldeath .......................................................... 71.1.1Caspases,themaineffectorsofapoptosis............................................................................ 71.1.2Theintrinsicpathway ............................................................................................................ 81.1.2.1TheBcl28family ............................................................................................................. 1.1.2.2Theapoptoticmitochondrialcascade ........................................................................... 91.1.2.3TheERstressapoptoticcascade ................................................................................... 91.1.3Theextrinsicpathway ......................................................................................................... 101.1.4Thetargetsofcaspases ....................................................................................................... 111.1.5Neuronalapoptosis ............................................................................................................. 111.1.5.1Apoptosisduringneuronaldevelopment.................................................................... 121.1.5.2Apoptosisinneurodegenerativediseases................................................................... 121.2Autophagy ............................................................................................................. 131.2.1Inductionandregulationofautophagy............................................................................... 131.2.2Theautophagicsequence.................................................................................................... 141.2.2.1RoleofATGsintheautophagosomeformation.......................................................... 141.2.2.2Theautophagolysosome ............................................................................................. 141.2.3Physiological15autophagy ...................................................................................................... 1.2.3.1Autophagicdegradationofcellularcomponents ........................................................ 151.2.3.2Autophagy:thetypeIIprogrammedcelldeath .......................................................... 151.2.4Autophagyinneuropathologies .......................................................................................... 161.3Interplaybetweenautophagicandapoptoticpathways......................................... 171.3.1Apoptosisblockadeinducesautophagy .............................................................................. 171.3.2Blockadeofautophagyinducesapoptosis .......................................................................... 171.3.3Crosstalkbetweenapoptosisandautophagy ..................................................................... 171.3.3.1RegulationofapoptosisbyATGs................................................................................. 171.3.3.2RegulationofautophagybyBcl2familymembersandcaspases .............................. 171.3.3.3Regulationofautophagybytheapoptoticextrinsicpathway .................................... 182.Themousecerebellum ........................................................................................ 192.1Thecerebellaranatomy ......................................................................................... 192.1.1General19organization ........................................................................................................... 2.1.2Thecerebellarcortex........................................................................................................... 192.1.2.1ThePurkinjecell .......................................................................................................... 192.1.2.2Thegranulecells.......................................................................................................... 202.1.2.3Theinterneurons......................................................................................................... 212.1.3Thecerebellarafferents ...................................................................................................... 212.1.3.1Theolivocerebellarsystem ......................................................................................... 212.1.3.2Themossyfiberrelaysystem ...................................................................................... 222.1.4Thecerebellarefferents ...................................................................................................... 232.1.5Thecerebellarcircuitry........................................................................................................ 242.1.6Developmentofthecerebellarneuronsandafferents....................................................... 24
2.1.6.1DevelopmentofthePurkinjecells .............................................................................. 252.1.6.2Developmentofthegranulecells ............................................................................... 262.1.6.3Developmentoftheclimbingfibers............................................................................ 262.1.6.4Developmentofthemossyfibers ............................................................................... 282.2Functionsofthe28cerebellum .................................................................................. 2.2.1Motorfunctions .................................................................................................................. 292.2.2Cognitivefunctions.............................................................................................................. 302.2.3Spatialfunctions .................................................................................................................. 303.ThehotfootandLurcherGrid2mutantmice ....................................................... 323.1TheGrid2gene ...................................................................................................... 32δ 3.2Structureandlocalizationof...................................................................... 32GluR 2 δ 3.2.1GluR 2isanorphanglutamatereceptor ............................................................................ 32δ 3.2.2TheGluR 2ofPurkinje33cells ................................................................................................ δ 3.3Molecularpartnersof................................................................................ 33GluR 2 δ 3.4GluR 2inLTD ........................................................................................................ 353.5Hotfoot,anaturalGrid2knockoutmouse............................................................. 353.5.1Thehotfootbehavioralphenotype ..................................................................................... 36ho/ho δ 3.5.2DevelopmentandsynaptogenesisofthePurkinjecellsinthehotfootGluR 2mice... 363.6TheLurchermutationkillsPurkinjecells ................................................................ 373.6.1TheLurchermutationandglutamatereceptors ................................................................. 373.6.2ThemolecularbasisofPurkinjecelldeathintheLurchermouse....................................... 384.TheNagasakiprionproteindeficientmice ......................................................... 404.1Theprionproteinfamily ........................................................................................ 404.1.1TheprionproteingenePrnp ............................................................................................... 404.1.2TheDoppelgene40Prnd ......................................................................................................... 4.1.3TheShadoogeneSprn......................................................................................................... 404.1.4Theprionproteins ............................................................................................................... 414.1.4.1Thecellularprionprotein............................................................................................ 414.1.4.2Doppel ......................................................................................................................... 414.1.4.3Shadoo......................................................................................................................... 41C 4.2ThecellularprionproteinPrP ............................................................................... 42C 4.2.1ExpressionofPrPinthecentralnervous42system ............................................................... C 4.2.2CelltraffickingofPrP .......................................................................................................... 43C 4.2.3NeuronaleffectsofPrPdeficiency..................................................................................... 43C 4.2.4Molecularpartnersof44PrP .................................................................................................. C 4.2.5Copperbindingandantioxidativepropertiesof45PrP ........................................................ C 4.2.6Antiapoptoticactivityof46PrP ............................................................................................. 4.3TheprionproteinparalogueDoppel ...................................................................... 474.3.1SomaticandgerminalexpressionofDpl ............................................................................. 484.3.2PhysiologicalfunctionsofDpl ............................................................................................. 484.3.3TheneurodegenerativephenotypeoftheNagasakimouse ............................................... 494.3.4NeurotoxicityofDoppel ...................................................................................................... 49Ν− ∆ 4.3.4.1DoppelcanbeconsideredasaterminaltruncatedPrP( PrP).............................. 494.3.4.2Proapoptoticpropertiesof50Doppel ............................................................................ C 4.3.4.3DoppelandPrPantagonism....................................................................................... 501.1.1.1.1Thecompetitionmodel .......................................................................................... 501.1.1.1.2Thesensitizationmodel.......................................................................................... 50
Results ....................................................................................................................... 521.Purkinjecelldeathmechanismsinducedbymutationsoftheglutamatergicδ GluR 2receptorinmouse .......................................................................................... 521.1Publication1.LurcherGRID2induceddeathanddepolarizationcanbedissociatedincerebellarPurkinjecells.SelimiF,LohofAM,HeitzS,LalouetteA,JarvisCI,BaillyY,MarianiJ.Neuron(2003)37:8139. .................................................................................... 52Lc δ 1.2GluR 2inducedexcitotoxicitykillsPurkinjecells ................................................. 60Lc/+ δ 1.2.1BlockadeofionfluxexcitotoxicityrescuesGluR 2Purkinjecells................................... 601.2.2BlockadeofionotropicexcitotoxicitysuppressesautophagyandrescuesdendriticLc/+ δ developmentofGluR 2Purkinjecells............................................................................................ 611.3ImpairedsurvivalanddendriticdevelopmentofhotfootPurkinjecellsexvivo. ..... 611.4Delayedclimbingfibertranslocationinthedevelopinghotfootcerebellarcortex .. 622.Doppelinducedcelldeathmechanism(s)inprionproteindeficientPurkinjecellsoftheNagasakimutant62mouse .................................................................................. 2.1Publication2.BaxcontributestoDoppelinducedapoptosisofprionproteindeficientPurkinjecells.HeitzS,ZanjaniH,LutzY,GautheronV,BombardeG,RichardF,FuchsJP,VogelM,MarianiJ,BaillyY.DevNeurobiol,(2007)67:670686. ......................... 632.2Publication3.BCL2counteractsDplinducedapoptosisofprionproteindeficient0/0PurkinjecellsintheNgskPrnpmouse.HeitzS,GautheronV,LutzY, RodeauJL,ZanjaniHS,SugiharaI,BombardeG,RichardF,FuchsJP,VogelMW,MarianiJ,BaillyY.DevNeurobiol,(2008)68:332348............................................................................................. 812.3Publication4.AutophagyandcelldeathofPurkinjecellsoverexpressingDoppelinNgskPrnpdeficientmice.HeitzS,LeschieraR,HaeberléAM,DemaisV,GrantN,BombardeG,BaillyY.BrainPathol,inreview. .................................................................................... 99Discussion .................................................................................................................1381.ExcitotoxicityandautophagyarerelatedduringLurcherPurkinjecelldeath .....1382.MultipledeathmechanismsinducedbyDplinNagasakiPurkinjecells ..............1413.DifferentialcombinationofapoptosisandautophagyinNagasakiandLurchercerebellarPurkinjecells.............................................................................................143MaterialandMethods ..............................................................................................1451.Animalsandgenotyping ....................................................................................1451.1Animals ............................................................................................................... 145Lc/+ ho/ho 1.1.1TheGrid2 (Lurcher)andtheGrid2(hotfoot)mice .................................................. 1450/0 0/0/0/0 1.1.2TheNP ,theNP :BaxandtheNPHubcl2mice ................................................... 1451.2Genotyping.......................................................................................................... 1461.2.1DNAextraction .................................................................................................................. 1461.2.2146Genotyping ........................................................................................................................ 2.Methods ............................................................................................................1482.1Organotypiccerebellarculture............................................................................. 1482.2Histology ............................................................................................................. 1482.3Immunohistochemitry ......................................................................................... 1482.3.1Tissuesections................................................................................................................... 148
2.3.2Immunohistochemistry ..................................................................................................... 1492.3.3Immunohistofluorescenceinorganotypiccerebellarcultures.......................................... 1492.4Transmissionelectronmicroscopy ....................................................................... 1502.5Westernblotting ................................................................................................. 1502.6Quantitativeanalysis ........................................................................................... 1502.6.1MorphometricanalysisofPurkinjecelldendritictreeinorganotypiccerebellarcultures 1502.6.2QuantitativeanalysisofPurkinjecellsinorganotypiccerebellarcultures........................ 151Appendix...................................................................................................................1521.ModelsofPurkinjecelldegeneration.................................................................1521.1Purkinjecelldeathandmurine152mutations ........................................................... 1.1.1Thenervousmutantmouse .............................................................................................. 1521.1.2Thetopplermutantmouse ............................................................................................... 1521.1.3ThePurkinjecelldegeneration(pcd)mutant153mouse ........................................................ 1.1.4Thewoozymutantmouse................................................................................................. 1531.1.5TheNiemannPickdiseasetypeC...................................................................................... 1531.1.6Theleanermutantmouse ................................................................................................. 1541.1.7Thehyperspinymouse ...................................................................................................... 1541.1.8Thetambaleantemouse ................................................................................................... 1541.1.9Theweavermutantmouse ............................................................................................... 1551.1.10Thestaggerermutant155mouse ............................................................................................ 1.1.11Thereelermutantmouse.................................................................................................. 1561.2Purkinjecelldeathinneurologicaldisorders ........................................................ 1561.2.1Brainischemia ................................................................................................................... 1561.2.2Alzheimerdisease.............................................................................................................. 1561.2.3Huntingtondisease ........................................................................................................... 1561.2.4Priondiseases.................................................................................................................... 1572.Priondiseases ....................................................................................................1572.1ThePrionConcept:aproteinonlyhypothesisofinfection ................................... 1582.2Molecularandcellularbasisofneurodegenerationinpriondiseases ................... 1582.2.1Apoptosis........................................................................................................................... 1582.2.2Autophagy ......................................................................................................................... 1592.2.3Synapticanddendriticpathology...................................................................................... 159References ................................................................................................................160Communications .......................................................................................................197
3-MA ABC Apaf1 ATG BH BSE CARD CJD DED DFF45 DISC DIV DNA Dpl E13.5 EGL ER ERAD FADD Fas FLIP GABA GluR ho IP3R IR JNK LTD LTP Lc LYATT MAPK MEM MOMP NASP NDUFS Neo NGS NHS ORF
Abbreviations
3-Methyladenine Avidine Biotin Complex Apoptosis protease activating factor-1 Autophagy gene Bcl homology Bovine spongiform encephalopathy Caspase recruitment domain Creutzfeld Jacob disease Death effector domain DNA fragmentation factor 45kDa Death-inducing signal complex Daysin vitro Desoxyribonucleic acid Doppel Embryonic day 13.5 External germinal layer Endoplasmic reticulum Endoplasmic reticulum associated degradation Fas-associated death domain Fibroblast-associated FADD-like-ICE-inhibitory protein γamino butyric acid Glutamate receptor hotfoot Inositol triphosphate receptor Immunoreactivity c-jun N-terminal protein kinase Long term depression Long term potentiation Lurcher Lysosomal amino acid transporter Mitogen-activated protein kinase Minimum essential medium Mitochondrial outer membrane permeabilisation 1-Naphtyl-acetyl-spermine Subunit of the mitochondrial respiratory complex I Neomycine Normal goat serum Normal horse serum Open reading frame
1
P2 PB PBS PCD PCR PI3-K PKC C PrP res PrP PSD PTP RML ROS rpm Scrg1 SOD SQSTM SSCP TNF-R1 TOR TRADD TRAIL-R TSE UPR
Postnatal day 2 Phosphate buffer Phosphate buffer saline Programmed cell death Polymerase chain reaction Phosphoinositide 3-kinase Proteine kinase C Cellular prion protein Prion protein resistant to proteinase K Post-synaptic density Protein tyrosine phosphatase Rocky mountain laboratory Reactive oxygen species Rotations per minute Scrapie responsive gene 1 Superoxide dismutase Sequestosome Single-strand conformation polymorphism Tumor necrosis factor receptor 1 Target of rapamycin TNF-R-associated death domain TNF-related apoptosis-inducing ligand receptorTransmissible spongiform encephalopathy Unfolded protein response
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Introduction
Introduction
During the course of my thesis, I investigated the participation of apoptotic and autophagic cell death programs in neuropathologies. Neuronal cell death mechanisms are known to play a major role in neurodegenerative diseases and the physiopathological significance of the interplay between apoptotic and autophagic cascades is still not understood. Insights into the complex patterns of neuronal cell death observed in nervous
system diseases are critically needed to take up the challenge of designing novel neurodegenerative disease therapies, specifically targeting cell death pathways. A million people worldwide are affected by neurodegenerative diseases, a heterogeneous group of degenerative conditions affecting specific areas of the central nervous system. The majority of neurodegenerative pathologies are age-related disorders, and these diseases are becoming an increasing health and socio-economical problem in industrialized countries (Mayeux, 2003). Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer, Parkinson, Huntington and prion diseases induce progressive cognitive or movement impairment depending on the type of neuronal cells undergoing selective degeneration (Troncoso et al., 1996; Cleveland, 1999; Nunomura et al., 2007). Although these diseases are phenotypically well described, the molecular mechanisms leading ultimately to neuronal death remain unclear, and despite vigorous research efforts, therapy options have not been found. In Alzheimer (Nakagawa et al., 2000; Nixon et al., 2005), Parkinson (Webb et al., 2003; Hayley et al., 2004), Huntington (Hickey and Chesselet, 2003) and prion (Lucassen et al., 1995; Liberski et al., 2008) diseases, autophagy and apoptosis have been shown to be activated in parallel. If apoptosis is an absolute programmed cell death mechanism, in most cases, autophagy constitutes cell defense mechanisms towards cellular dysfunction or stress. Thus, both apoptosis and autophagy may be triggered by common upstream
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