2011.10.15.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Peter Pazmany Catholic University Faculty of Information Technology BEVEZETÉS A FUNKCIONÁLIS NEUROBIOLÓGIÁBA INTRODUCTION TO FUNCTIONAL NEUROBIOLOGY www.itk.ppke.hu By Imre Kalló Contributed by: Tamás Freund, Zsolt Liposits, Zoltán Nusser, László Acsády, Szabolcs Káli, József Haller, Zsófia Maglóczky, Nórbert Hájos, Emilia Madarász, György Karmos, Miklós Palkovits, Anita Kamondi, Lóránd Erőss, Róbert Gábriel, Zoltán Kisvárday Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 www.itk.ppke.hu Stem cells and potential applicationsImre Kalló & Emilia MadarászPázmány Péter Catholic University, Faculty of Information Technology I. Undifferentiated cells during embryogenesis and throughout life II. Proliferation versus differentiation III. Migration, axonal growth and synapse formation IV. Potential application of stem cells; from experiments to therapy Introduction to functional neurobiology: Stem cells J_GABA_N3_24.jpg 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 www.itk.ppke.hu Neurons maintain a complex cellular morphology Axon Dendrite kosarsejt2meretforgatott 100 µm s.r. s.o. s.p. Dendrite (D) Cell body Axon (A) Pyramidal cell Cerebral cortex Interneuron Hippocampus D A Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 www.itk.ppke.hu Two dogma persisted about neurons for a long time 1. Mature neurons do not divide: 2. We have to manage our life with the set of neurons present at early postnatal period: 1984 Notebohm: song-birds 1992 Reinolds és Weiss: rodents, human Conclusion of these studies: Nondifferentiated cells with the capability to produce new neurons are present throughout life. Where are the neurogenic zones? Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 www.itk.ppke.hu Location of proliferating cells (forming a germinative layer) in the neural primordium of human and rat embryos Theneuralplatecomprisestheshoehorn-shapeddorsalthickeningoftheectodermattherostralendoftheearlyembryo.Theneuralplateformstheneuraltube,whichinitiallyremainsopenatthecaudalandrostralends.Therostralpartoftheneuraltubeestablishestheprimordiumofthebrain.Theneuraltubeiscomposedofasinglelayerofproliferatingcells,theprimarygerminativelayer.Asecondgerminativelayerappearsinlaterstagesofembryonicdevelopmentundertheventricularwallthrough-outtheentireneuraxis.Intheventralpartoftheforebrainthesubventricularzoneisthebestexploredresidenceofthestemcellsduringadulthood.Similarimportantadultneurogeniczone,thesubgranularzoneislocatedbeneaththegranulecelllayerofthehippocampus. Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 www.itk.ppke.hu Location of proliferating cells (forming a germinative layer) in the neural primordium of human and rat embryos 6-day old human embryo 8 –day old human embryo Rat forebrain Primary germinative zone Secundary germinative zone E10.5 Adult Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 www.itk.ppke.hu Location of stem cells (with the capability to proliferate) in the adult rat brain Subventricular zone Subgranular zone Cerebral cortex Olfactory bulb Ventricle Cerebellum Brainstem Corpuscallosum Primary migratory pathway Thalamus Neurosphere From a new born mouse forebrain 10µm Hippocampus Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 www.itk.ppke.hu Cell proliferation and interkinetic migration of the cell nucleus in the primary ventricular zone + + Location of future pial surface G1 S M Cell-phase Location of future ventricular surface Symmetrical mitosis Asymmetrical mitosis Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 www.itk.ppke.hu Cell proliferation and interkinetic migration of the cell nucleus in the primary ventricular zone ventricle Pia -ECM Radialglia Radial neuro-epithelial cell ECM Neuronal precursor Lateral induction / inhibition symmetric asymmetric mitoses Notch Hes ErbB2 BLBP Delta-1 Mash/Ngn1,2 NRG-1 Ref: HalfterW, Dong S, Yip YP, Willem M, Mayer U. A critical function of the pialbasement membrane in cortical histogenesis. J Neurosci. 2002 Jul 15;22(14):6029-40. Radial glia.jpg Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 www.itk.ppke.hu Notch/Delta system notch notch differentiating cell undifferentiating cell In general: Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 www.itk.ppke.hu Signalling pathways activated DSL –Delta, Serrate, Lag-2 CSL –CBF1, Su(H), Lag-1 NICD –Notch Intracellular Domain TACE/ADAM17 –TNF.Converting Enzyme ADisintegrin And Metalloprotease Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 www.itk.ppke.hu Lateral inhibition (Drosophila proneuronal cluster) Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 www.itk.ppke.hu Role of bHLH transcription factors during differentiation Basic helix-loop-helix transcriptional factors H bHLH L H Pro-neural Mash 1..., Ngn 1,2, Math 1 Neural Math 2, NeuroD RyoichiroKageyamaand ShigetadaNakanishi; Current Opinion in Genetics & Development 1997, 7:659-665 Neural progenitor Neural precursor Neuron Notch Delta Notch ICD RBP-J Hes1 Hes5 Mash1 Math1 Ngn E2A NeuroD Math2 E2A Hes1 Hes5 Mash1 Math1 ngn NeuroD Math2 Neuron-specific genes Cytoplasm Nucleus Groucho Migration Terminal differentiation Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 www.itk.ppke.hu Migration of embryonic cells from the primary germinative zone Rakic2 Radialglia Migrating neuronal precursors RakicP. J. Comp. Neurol. 1972, 145: 61-84 bhg018h5 Neuronal precursors deriving from the primary germinative zone migrate along the processes of radial glial cells Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 www.itk.ppke.hu The secundary germinative zone derives from the primary one: Primarygerminativelayer Secundary germinative layer prenatal postnatal Wide upward diagonal Projecting neuron Diagonal brick Small projecting neuron Zsindely Zsindely Localinterneuron Astroglial cells Nagy konfetti Radialglia Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Nagy konfetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Large confetti Sphere Sphere Sphere Sphere Sphere Sphere Sphere ependyma VZ SVZ Projecting neurons are not produced The neurogenic zones in adulthood are remnants of the SVZ + dentate gyrus subgranular zone (SGZ) Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 www.itk.ppke.hu Migration of cells in adulthood Large confetti brain tissue ependyma ventricle The neuronal precursors migrate among the already differentiated hippocampal neurons The neuronal precursors migrate separated from the already differentiated neurons Subgranular zone Subventricular zone Doetsch F et al. J. Neurosci. 1997, 17(13): 5046 ; Sanai et al. Nature 2004, 427: 740 Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 www.itk.ppke.hu The antero-posterior axis is defined by the primitive streak and antero-visceral entoderm (AVE) epiblast hypoblast AVE Primitivestreak A P testtengely AVE Primitívárok A P testtengely Nodus Schönwolf, Colas; DEVELOPMENTAL DYNAMICS 221:117–145 (2001) Primitive groove Body axis Body axis Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 www.itk.ppke.hu The antero-posterior axis is defined by the primitive streak and antero-visceral entoderma (AVE) Dorso-ventral axis: Shh / Wnt P A L L M Shh BMP BMP A Light horizontal BMP, Wnt, Zic, Gli3 neural crest SHH alarplate basalplate Gli 1, 2; Nkx; Dlx Pax Emx2 roof plate basal plate SHH, Gli1,2 B A P V D Shimamura, Rubenstein; 1995 Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 www.itk.ppke.hu Cranio-caudal expression pattern of homeobox genes 14f06 Hirthet al., (1998). Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 www.itk.ppke.hu Differentiation of rhombomeres Dev_Brainves_1.jpg Dev_Brainves_2.jpg Dev_Brainves_3.jpg Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 www.itk.ppke.hu Differentiation of neuronal populations in the brain nrn0201_099a_f4 Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 www.itk.ppke.hu The cerebral cortex is built up by cells deriving from different regions of both the primary and secundary germinative layers E10.5 E10,5 E1 0, 5 E1 0,5 E10, 5 Adult Neuron kép Shuurmans 2004 Alvarez-Buylla 1998 interneuron migráció 2 Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 www.itk.ppke.hu Developing of neuronal pathways: axonal growth and path finding growth cone Phase-contrast image of growth cone Electron microscopic image of growth cone Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 www.itk.ppke.hu Burnette et al., 2008 Cell Developing of neuronal pathways: axonal growth and pathfinding Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 www.itk.ppke.hu „Pathfinder” processes: directed growth and „forbidden” directions Timed appearance of guiding molecules and receptors! Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 www.itk.ppke.hu Ideal adherence of the growth cone to the surface of neighboring processes; they form bundles Molnár Zoltán felvétele Processes of cortical neurons„guide” the processes of thalamic neurons to the cortex Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 www.itk.ppke.hu Axonal pathfinding in the visual pathway VisualPathway1.jpg VisualPathway2.jpg VisualPathway3.jpg Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 www.itk.ppke.hu „Fire together –wire together” Bioelectric activity [Ca2+]I Release of materials BDNF GABA [Ca2+]I Dark horizontal phosphorilation Protein synthesis Release of signal molecules Dark horizontal Dark horizontal ECM Protein synthesis and release Growth cone Target cell GABAA GABAB Synapse_Nature_2 GDP Jelitai, Madarasz; Int.Rev.Neurobiol. 2006 Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 www.itk.ppke.hu Formation and subsistence of functional neuronal networks is the result of multiple selection Production of cells: surviving cells are selected from a multitude of cells with great surplus Migration of cells: cells are selected by permissive, attractive and repulsive adhesive signals Axonal growth/withdrawal: the axonal sprouting is excessive; sustained axons „are being selected” by the activity they show Synapse formation/survival: it is possible only among mutually active partners Selection by the ambience Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 www.itk.ppke.hu For what purpose can the stem cells be used? Today „only” for scientific/clinical research Stem cell therapy is still not sufficiently validated for neuronal diseases and currently is not a choice……. But there are clinical studies with controls! (Must be free of charge for the patients!!!) Secernated factors inducing regeneration, and/or reducing inflammation? Gene-therapy Clinical trials: LSDs(e.g. Batten-disease) Future: Great possibilities in stem cell therapy Introduction to functional neurobiology: Stem cells 2011.10.15. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 www.itk.ppke.hu Location of proliferating cells (forming a germinative layer) in the neural primordium of human and rat embryos 6-day old human embryo 8 –day old human embryo Rat forebrain Primary germinative zone Secundary germinative zone E10.5 Adult