REGULATION OF VERTEBRATE NEURON DEATH IN DEVELOPMENT AND NEURODEGENERATION: ROLE OF TRANSCRIPTION LLOYD A. GREENE COLUMBIA UNIVERSITY NEW YORK, NY

MASSIVE LOSS OF NEURONS DURING VERTEBRATE DEVELOPMENT HAS BEEN LONG KNOWN BEARD (1889) - LOSS OF NEURONAL POPULATIONS IN FISH (ROHON-BEARD NEURONS) COLLIN (1906) - DEATH OF MANY SENSORY AND MOTOR NEURONS IN THE CHICK EMBRYO

APPROXIMATELY 50% OF POST-MITOTIC NEURONS DIE DURING NORMAL DEVELOPMENT NEURON NUMBER IN CHICK ION 25,000 20,000 15,000 10,000 5,000 0 Clarke, Rogers & Cowan J. Comp. Neurol. 167: 125 (1976) NORMAL 36 38 40 42 44 46 P2 P4 DEVELOPMENTAL STAGE

APOPTOTIC NEURONAL DEATH IN DEVELOPING SUBSTANTIA NIGRA R. Burke Cell Tiss Res (2004)

DEATH OF PROLIFERATING VZ CELLS IN E14 MOUSE CEREBRUM Tan = BrDU; Purple = ISEL/dying Blaschke et al J Comp Neurol 1998

REGULATED DEVELOPMENTAL DEATH OF OLIGODENDROCYTES OLIGO SURVIVAL 100 90 80 70 60 50 40 30 20 10 0 GENERATED SURVIVI OLIGOS 3 DAYS FROM Barres and Raff, Journal of Cell Biology, Volume 147, Number 6, December 13, 1999 1123-1128

VIKTOR HAMBURGER AND RITA LEVI-MONTALCINI AND THE BEGINNING OF THE NEUROTROPHIC THEORY OF DEVELOPMENTAL NEURON DEATH IN VERTEBRATES

VICTOR HAMBURGER AND THE NOTION THAT PERIPHERAL TARGETS REGULATE NEURONAL CELL DEATH

ELMER BEUKER AND THE EFFECTS OF SARCOMA 180 ON NEURONAL SURVIVAL

LEVI-MONTALCINI AND THE DISCOVERY OF A DIFFUSIBLE SURVIVAL-PROMOTING ACTIVITY

LEVI-MONTALCINI AND HAMBURGER DISCOVER A SOLUBLE NERVE-GROWTH PROMOTING ACTIVITY (NGF) IN EXTRACTS OF SARCOMA 180 SARCOMA EXTRACT

PURIFIED NGF PROMOTES NEURON SURVIVAL IN VITRO NGF

NGF PROMOTES SYMPATHETIC & SENSORY NEURON SURVIVAL IN VITRO -NGF +NGF

EVIDENCE FOR THE ROLE OF GROWTH FACTORS IN REGULATING DEVELOPMENTAL NEURONAL DEATH IN VIVO 1. INJECTION OF NEW BORN MICE WITH NGF ANTIBODIES YIELDS IMMUNOSYMPATHECTOMY MOTHERS AUTOIMMUNIZED AGAINST NGF PRODUCE OFFSPRING WITH MANY FEWER DORSAL ROOT GANGLIONIC NEURONS 2. SUPPLY OF EXOGENOUS NGF BLOCKS DEVELOPMENTAL CELL DEATH OF SYMPATHETIC NEURONS

EVIDENCE FOR THE ROLE OF GROWTH FACTORS IN REGULATING DEVELOPMENTAL NEURONAL DEATH IN VIVO MICE NULL FOR NGF OR ITS RECEPTORS SHOW EXCESS NEURONAL DEATH. Fagin et al. J Neurosci (1996)

NORMAL DEVELOPMENTAL NEURONAL DEATH OCCURS AND IS REGULATED BY TARGET DERIVED TROPHIC FACTORS NEURON NUMBER IN CHICK ION 25,000 20,000 15,000 10,000 5,000 0 NORMAL ENUCLEATED 36 38 40 42 44 46 P2 P4 DEVELOPMENTAL STAGE Clarke, Rogers & Cowan J. Comp. Neurol. 167: 125 (1976)

RETROGRADE TRANSPORT OF NEUROTROPHINS FROM TARGETS 125 I-NGF SCG CPM In SCG 450 400 350 300 250 200 150 100 50 0 0 8 16 24 32 40 48 HOURS CONTRATERAL IPSILATERAL Ian Hendry & Hans Thoenen

NEURONAL SURVIVAL AS REGULATED BY COMPETITION FOR TARGET-SUPPLIED TROPHIC FACTOR

ALL TROPHIC SUPPORT DURING DEVELOPMENT IS NOT NECESSARILY TARGET DERIVED - WHAT KEEPS NEURONS ALIVE BEFORE THEY INTERACT WITH THEIR TARGETS? AUTOCRINE LOOPS -NEURONS CAN MAKE THEIR OWN NEUROTROPHIC FACTORS (CORTICAL NEURONS; BDNF) TISSUE EN ROUTE TO TARGET MAY PROVIDE TROPHIC FACTORS (SUCH AS NT3 AND BDNF)

GROWTH FACTORS THAT CAN MAINTAIN SURVIVAL OF SELECT NEURONAL POPULATIONS NEUROTROPHINS NGF BDNF NT3 NT4/5 EGF FAMILY EGF TGFα NEUREGULINS FGF FAMILY INSULIN & IGFs TGFβ SUPERFAMILY TGFβ BMPs GDNF ARTEMIN NURTURIN PERSEPHIN CYTOKINES CNTF LIF INTERLEUKINS PDGF HGF FAMILY HGF MSP NEUROPEPTIDES PACAP VIP

REGULATED DEVELOPMENTAL DEATH OF OLIGODENDROCYTES NEUREGULIN, PDGF 100 90 80 70 60 50 40 30 20 10 0 GENERATED SURVIVI OLIGOS 3 DAYS FROM Barres and Raff, Journal of Cell Biology, Volume 147, Number 6, December 13, 1999 1123-1128

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? REMOVAL OF NEURONS WITH TRANSIENT FUNCTIONS ROHON-BEARD CELLS - LOSS IN ZEBRA FISH Williams et al. Dev Biol (2000)

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? CREATION OF SEXUALLY DIMORPHIC STRUCTURES

SEXUALLY DIMORPHIC NEURON DEATH IN RAT BSTpr - REGULATION BY TESTOSTERONE FEMALES MALES MALES FEMALES POST NATAL DAY EX-MALES BSTpr =Principal nucleus of the Bed nucleus of the striatus terminalis M & EX-M + TEST Chung et al. J Neurbiol (2000)

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? ELIMINATION OF ECTOPIC NEURONS

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? ELIMINATION OF NEURONS WITH ECTOPIC PROJECTIONS

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? SYSTEMS MATCHING: CREATION OF OPTIMAL LEVELS OF INNERVATION BETWEEN INTERCONNECTED GROUPS OF NEURONS AND BETWEEN NEURONS AND THEIR NON-NEURONAL TARGETS (Buss and Oppenheim Anat Sci Int, 2004) WHAT HAPPENS IF DEVELOPMENTAL NEURON CELL DEATH IS ABSENT?

IN ABSENCE OF NORMAL DEVELOPMENTAL CELL DEATH MOST EXCESS NEURONS BECOME ATROPHIC: EXAMPLE OF FACIAL NERVE White et al. J Neurosci 1998

RETINAL NEURON RESPONSES TO LIGHT CHANGE IN ABSENCE OF NORMAL DEVELOPMENTAL CELL DEATH 450 400 350 300 250 200 150 100 50 WT BAX-/- mv Relative number of responding neurons GCL=ganglion cell layer INL=inner nuclear layer ONL=outer nuclear layer IPL=inner plexiform layer 0 ROD TEST CONE TEST (Péquignot et al. Dev Dyn 2003

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? PREVENT OVER-PRODUCTION OF NEUROPROGENITOR CELLS TO GENERATE PROPER SIZED BRAIN

EMBRYOGENIC DEFECTS IN A MOUSE LACKING CASPASE-9 From: Kuida et al Cell:94: 325-337, 1998

WHY DOES CELL DEATH OCCUR IN NEURODEVELOPMENT? ELIMINATION OF CELLS WITH DAMAGED DNA OR WITH OTHER DEFECTS

WHY DOES CELL DEATH OCCUR IN VERTEBRATE NEURODEVELOPMENT? A MEANS OF EVOLUTIONARY CHANGE: ADAPTIVE CHANGES IN THE ONTOGENETIC DEATH AND SURVIVAL OF CELLS IN REPSPONSE TO GENETIC MUTATION (E.G. THE PRODUCTION OF EXCESS NEURONS COULD BE USED FOR THE INNERVATION OF NEW TARGETS MADE AVAILABLE BY THE EVOLUTION OF LIMBS) (Buss and Oppenheim Anat Sci Int, 2004)

WHAT ARE THE MECHANISMS BY WHICH NEURON SURVIVAL AND DEATH ARE REGULATED DURING VERTEBRATE DEVELOPMENT?

SPECIFICITY OF NEUROTROPHIN FAMILY RECEPTOR BINDING NGF NT3 BDNF NT4/5 TRKA TRKC TRKB

TRK SIGNALING AND PROMOTION OF NEURON SURVIVAL NGF TRKA WORTMANNIN LY2942002 D/N PI3K ACT-PI3K D/N AKT ACT-AKT PI3K AKT RAS ERK D/N-RAS ACT-RAS PD98059 U0126 DEATH IN SOME PARADIGMS SURVIVAL

EXAMPLES OF HOW AKT PROMOTES SURVIVAL BAD AKT P-BAD 14-3-3 (cytoplasmic) mitochondria DEATH FRKH AKT P-FRKH 14-3-3 (cytoplasmic) nucleus Bim DEATH TRANSCRIPTIONAL IAP AKT ACTIVATION IAP

MITOCHONDRIA AND APOPTOTIC DEATH APOPTOTIC STIMULI BAX BIM BAX BIM BIM BIM BCL2 mitochondrion BCL2 BAX BAX BAK EGL1 CED9 CED3 CYTOCHROME C APAF1 (APOPTOSOME) CED4 CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION

Plotted from data in: Yaginuma et al. J Neurosci 1996 DEVELOPMENTAL NEURON CELL DEATH IN VERTEBRATES REQUIRES MACROMOLECULAR SYNTHESIS 35 30 % PYKNOTIC CELLS IN CERVICAL SPINAL CORD OF CHICK EMBRYO 25 20 15 10 5 0 VEHICLE CYCLOHEX ACT-D

TRANSCRIPTIONAL REGULATION OF APOPTOTIC DEATH APOPTOTIC STIMULI P53 P63 p73 cjun FKH E2F NFKB BCL2 BIM BIM mitochondrion BAX BCL2 BAX AIF CYTOCHROME C APAF1 SMAC/DIABLO CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION IAPs

AKT BLOCKS DEATH AT MULTIPLE LEVELS OF THE APOPTOTIC MECHANISM APOPTOTIC STIMULI NEUROTROPHIC FACTORS/AKT P53/ P63/ P73 BAD FAS-L cjun FKH E2F NFKB FAS BIM BCL2 BAX BIM BCL2 BAX mitochondrion AIF CYTOCHROME C APAF1 BCLX-L SMAC BLOCK APOPTOTIC TRANSC. PATHWAYS (FKH PHOSPHORYLATION) ELEVATE ANTI- APOPTOTIC MOLECULES CASPASE 9 ACTIVATION CASPASE 3,6,7 ACTIVATION IAPs PHOSPHORYLATE, EXCLUDE PRO- APOPTOTIC BAD

C-JUN IS PHOSPHORYLATED/ACTIVATED IN NGF-DEPRIVED NEURONS Eilers et al. J Neurosci 1998

BLOCKING JNK ACTIVATION BLOCKS NEURON DEATH CAUSED BY NGF DEPRIVATION +NGF -NGF -NGF +CEP1347

DEATH SIGNALING BY THE JNK PATHWAY IN NEURON DEATH APOPTOTIC STIMULI PO4 GTP GDP RAC1 PO4 PO4 MLK MLK POSH PO4 MKK4/7 JNK JIP PO4 JUN BIM

BIM IS A PRO-APOPTOTIC MEMBER OF THE BCL2 FAMILY Puma

BIM IS UPREGULATED BY NGF DEPRIVATION, PARTLY VIA THE JNK PATHWAY AND CONTRIBUTES TO NEURON DEATH Bim = d/n Jun Whitfield et al Neuron 2001

MLKS AND POSH ARE DEGRADED IN VIABLE NEURONS APOPTOTIC STIMULI GDP RAC1 MLK MLK POSH MKK4/7 JNK JIP JUN BIM PROTEASOME

MLKS AND POSH ARE STABILIZED BY A FEEDBACK LOOP MECHANISM IN RESPONSE TO APOPTOTIC STIMULI APOPTOTIC STIMULI PO4 GTP RAC1 PO4 PO4 MLK MLK POSH PO4 MKK4/7 JNK JIP JUN BIM PROTEASOME

DEATH SIGNALING BY THE JNK PATHWAY IN NEURON DEATH APOPTOTIC STIMULI Hr -NGF 0 4 8 POSH PO4 JNK 1 2 3 Neuronal PC12 cells GTP RAC1 PO4 PO4 MLK MLK POSH PO4 MKK4/7 JNK JIP PO4 JUN BIM PROTEASOME

JIP AND POSH RELOCALIZE IN RESPONSE TO APOPTOTIC STIMULI i CONTROL DNA DAMAGE ii JIP iii iv v vi POSH

CELL CYCLE MOLECULES MEDIATE NEURON CELL DEATH EVOKED BY LOSS OF TROPHIC SUPPORT

E2F REGULATES CELL PROLIFERATION PO4 E2F Rb TK C D 1 CDK4 BLOCKED ACTIVATION TRANS-ACTIVATION PO4 Rb S-PHASE E2F CHR MOD C-MYB GENE REPRESSION GENE DE-REPRESSION

E2F REGULATES CELL PROLIFERATION & NEURON DEATH PO4 S-PHASE E2F1 Rb CHR MOD C-MYB C D 1 CDK4 GENE REPRESSION GENE DE-REPRESSION PO4 p130 CHR MOD E2F4 C-MYB GENE REPRESSION GENE DE-REPRESSION

APOPTOTIC STIMULI ACTIVATE CDK4/6 IN NEURONS

p130 IS THE MAJOR E2F PARTER ON CHROMATIN IN NEURONS, IS PHOSPHORYLATED AND LOST WITH NGF DEPRIVATION, RESULTING IN MYB DE-REPRESSION AND UP-REGULATION Antibody Control p130 p107 prb SCG NGF W/D Hrs 0 16 16 +F C-MYB +NGF p130 C- MYB -NGF

E2F REGULATES CELL PROLIFERATION & NEURON DEATH PO4 C D 1 CDK4 E2F4 p130 CHR MOD C-MYB GENE REPRESSION GENE DE-REPRESSION

THE E2F PATHWAY LEADS TO BIM UPREGULATION VIA CDK4 AND MYB A. Neuronal PC12 cells GFP Bim merge sibim sib-myb sic-myb sicdk4 siluc

E2F AND NEURON CELL DEATH PO4 C D 1 C CDK4 D 1 CDK4 E2F4 p130 SUVH39 HDAC GENE REPRESSION GENE DE-REPRESSION C-MYB APOPTOTIC STIMULI PO4 C-MYB C-JUN FOXO BIM

THE CDK4-E2F-p130-MYB-BIM PATHWAY OF NEURON CELL DEATH PO4 C D 1 CDK4 E2F4 p130 HDAC SUVH39 C-MYB GENE REPRESSION GENE DE-REPRESSION APOPTOTIC STIMULI PO4 C-MYB C-JUN FOX O BIM

THERE IS MASSIVE DEATH OF NEURONS, NEUROPROGENITORS AND OLIGODENDROGLIA IN NORMAL VERTEBRATE DEVELOPMENT THIS IS LARGELY REGULATED BY ACCESS TO LIMITING SUPPLIES OF EXOGENOUS SURVIVAL-PROMOTING TROPHIC FACTORS SURVIVAL IS PROMOTED LARGELY BY ACTIVATION OF AKT AS WELL AS OF ERKS AND INVOLVES BLOCKADE OF DEATH PATHWAYS AT MULTIPLE POINTS DEVELOPMENTAL NEURON DEATH IS TRANSCRIPTION DEPENDENT INDUCTION OF DEATH INVOLVES MULTIPLE PRO-APOTOTIC SIGNALING PATHWAYS, SOME OF WHICH CONVERGE ON INDUCTION OF BH3-DOMAIN PROTEINS