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Lecture V. Cell Birth and Death Bio 3411 Wednesday September 15, 2010 Lecture V. Lecture V. Cell Birth and Death Bio 3411 Wednesday September 15, 2010 Lecture V. Cell Birth & Death 1

Readings NEUROSCIENCE: 4 th ed, pp 596 -609 (end of Chapter 23: Construction of Readings NEUROSCIENCE: 4 th ed, pp 596 -609 (end of Chapter 23: Construction of Neural Circuits) References posted: • †Abbott, A. (2009). Neuroscience: One hundred years of Rita. Nature, • †Berry, D. (2006). Apoptosis and signal transduction. http: //www. wehi. edu. au/education/wehi 458(7238), 564 -567. -tv/? page=2. • †Cohen, S. (1987). Autobiography. http: //nobelprize. org/nobel_prizes/medicine/laureates/1986/cohen-autobio. html • †Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature, • †Hollyday, M. (2001). Viktor Hamburger (1900 -2001): A rememberance. Developmental 407(6805), 770 -776. Biology, 236(1), 1 -2. • †Raff, M. (1998). Cell suicide for beginners. Nature, 396(6707), 119 -122. ___________ †(pdfs on course websites: [http: //artsci. wustl. edu/~bio 3411/] & [http: //www. nslc. wustl. edu/courses/Bio 3411/bio 3411. html]) September 15, 2010 Lecture V. Cell Birth & Death 2

Cited A • • • Acehan, D. , Jiang, X. , Morgan, D. G. Cited A • • • Acehan, D. , Jiang, X. , Morgan, D. G. , Heuser, J. E. , Wang, X. , & Akey, C. W. (2002). Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell, 9(2), 423 -432. Brenner, S. (1973). The genetics of behaviour. Br Med Bull, 29(3), 269 -271. Cleary, M. L. , Smith, S. D. , & Sklar, J. (1986). Cloning and structural analysis of c. DNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14; 18) translocation. Cell, 47(1), 19 -28. Cohen, S. , Levi-Montalcini, R. , & Hamburger, V. (1954). A Nerve Growth-Stimulating Factor Isolated from Sarcom as 37 and 180. Proc Natl Acad Sci U S A, 40(10), 1014 -1018. Cowan, W. M. , & Wenger, E. (1967). Cell loss in the trochlear nucleus of the chick during normal development and after radical extirpation of the optic vesicle. J Exp Zool, 164(2), 267 -280. Ellis, R. E. , & Horvitz, H. R. (1991). Two C. elegans genes control the programmed deaths of specific cells in the pharynx. Development, 112(2), 591 -603. Ellis, R. E. , Yuan, J. Y. , & Horvitz, H. R. (1991). Mechanisms and functions of cell death. Annu Rev Cell Biol, 7, 663 -698. Fesik, S. W. (2000). Insights into programmed cell death through structural biology. Cell, 103(2), 273 -282. Gross, A. , Mc. Donnell, J. M. , & Korsmeyer, S. J. (1999). BCL-2 family members and the mitochondria in apoptosis. Genes Dev, 13(15), 1899 -1911. Haldar, S. , Reed, J. C. , Beatty, C. , & Croce, C. M. (1990). Role of bcl-2 in growth factor triggered signal transduction. Cancer Res, 50(22), 7399 -7401. September 15, 2010 Lecture V. Cell Birth & Death 3

Cited B • • • Hamburger, V. (1958). Regression versus peripheral control of differentiation Cited B • • • Hamburger, V. (1958). Regression versus peripheral control of differentiation in motor hypoplasia. Am J Anat, 102(3), 365 -409. Hamburger, V. (1975). Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol, 160(4), 535 -546. Hamburger, V. (1977). The developmental history of the motor neuron. Neurosci Res Program Bull, 15 Suppl, iii-37. Kerr, J. F. , Wyllie, A. H. , & Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 26(4), 239 -257. Korsmeyer, S. J. (1992). Bcl-2: an antidote to programmed cell death. Cancer Surv, 15, 105 -118. Landmesser, L. , & Pilar, G. (1974). Synaptic transmission and cell death during normal ganglionic development. J Physiol, 241(3), 737 -749. Levi-Montalcini, R. , & Hamburger, V. (1951). Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J Exp Zool, 116(2), 321 -361. Levi-Montalcini, R. , Meyer, H. , & Hamburger, V. (1954). In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo. Cancer Res, 14(1), 49 -57. Schlesinger, P. H. , Ferdani, R. , Liu, J. , Pajewska, J. , Pajewski, R. , Saito, M. , Shabany, H. , & Gokel, G. W. (2002). SCMTR: a chloride-selective, membrane-anchored peptide channel that exhibits voltage gating. J Am Chem Soc, 124(9), 1848 -1849. Sulston, J. E. , & Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol, 56(1), 110 -156. September 15, 2010 Lecture V. Cell Birth & Death 4

Cited C • • Sulston, J. E. , Schierenberg, E. , White, J. G. Cited C • • Sulston, J. E. , Schierenberg, E. , White, J. G. , & Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol, 100(1), 64 -119. Vaux, D. L. , Cory, S. , & Adams, J. M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature, 335(6189), 440 -442. White, J. G. , Horvitz, H. R. , & Sulston, J. E. (1982). Neurone differentiation in cell lineage mutants of Caenorhabditis elegans. Nature, 297(5867), 584 -587. Wiesmann, C. , Ultsch, M. H. , Bass, S. H. , & de Vos, A. M. (1999). Crystal structure of nerve growth factor in complex with the ligand-binding domain of the Trk. A receptor. Nature, 401(6749), 184 -188. Xue, D. , & Horvitz, H. R. (1997). Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature, 390(6657), 305 -308. Xue, D. , Shaham, S. , & Horvitz, H. R. (1996). The Caenorhabditis elegans cell-death protein CED -3 is a cysteine protease with substrate specificities similar to those of the human CPP 32 protease. Genes Dev, 10(9), 1073 -1083. Yang, X. , Chang, H. Y. , & Baltimore, D. (1998). Essential role of CED-4 oligomerization in CED-3 activation and apoptosis. Science, 281(5381), 1355 -1357. Yuan, J. Y. , & Horvitz, H. R. (1990). The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev Biol, 138(1), 33 -41. September 15, 2010 Lecture V. Cell Birth & Death 5

What the last Lecture was about • • Mesoderm induces neuroectoderm in overlying ectoderm What the last Lecture was about • • Mesoderm induces neuroectoderm in overlying ectoderm that gives rise to neuronal or epidermal cells. The “default” state of neuroectodermal cells is neuronal. Neuroectoderm secretes Bone Morphogenic Protein-4 (BMP-4), a signaling molecule that blocks the neuronal fate in neighboring neuroectodermal cells. Mesoderm secretes proteins - Chordin, Noggin, Follistatin - that block BMP 4 and neuroectodermal cells continue as neuronal progenitors. This inductive mechanism is conserved between vertebrates and invertebrates. These, and other similar, signaling mechanisms are used by the developing nervous system to control other events later in development. BMP-4 is a member of the Transforming Growth Factor-beta (TGF-β) family of signaling molecules. September 15, 2010 Lecture V. Cell Birth & Death 6

What this Lecture is about • Cell Death – Necrosis vs Apoptosis • Promoting What this Lecture is about • Cell Death – Necrosis vs Apoptosis • Promoting growth and survival – “trophism” • Inhibition of the “death mechanism” • Broader implications: neuroembryology; cancer • Different critters - Same genes • Molecular models • Connection of Trophic Factors to cell death September 15, 2010 Lecture V. Cell Birth & Death 7

Apoptosis Kerr, J. F. , et al. , (1972) September 15, 2010 from Greek Apoptosis Kerr, J. F. , et al. , (1972) September 15, 2010 from Greek “apo” meaning “separation” & “ptosis” for “falling off” Lecture V. Cell Birth & Death 8

Types of Cell Death • Not Self-Initiated Necrosis (Provoked) Apoptosis (Programmed) • Not Stereotypic Types of Cell Death • Not Self-Initiated Necrosis (Provoked) Apoptosis (Programmed) • Not Stereotypic • Can Be Slow • “Messy” (injury can spread) • Cell-Autonomous • Stereotypic • Rapid • “Clean” (dead cells eaten) Ellis, R. E. , et al. , (1991) September 15, 2010 Lecture V. Cell Birth & Death 9

Removing a neuron’s targets, leads to its death Hamburger, V. (1958, 1977) September 15, Removing a neuron’s targets, leads to its death Hamburger, V. (1958, 1977) September 15, 2010 Deprived Lecture V. Cell Birth & Death Normal 10

Neuronal death is central for normal NS development Hamburger, (1975); Landmesser & Pilar, (1974); Neuronal death is central for normal NS development Hamburger, (1975); Landmesser & Pilar, (1974); Cowan & Wenger, (1967) September 15, 2010 Lecture V. Cell Birth & Death 11

Neuron survival correlates with target innervation Motor neurons Target Muscles Axon Outgrowth Development Progresses Neuron survival correlates with target innervation Motor neurons Target Muscles Axon Outgrowth Development Progresses Target Innervation Not all neurons innervate targets Neuronal Loss September 15, 2010 Lecture V. Cell Birth & Death 12

Target innervation determines which neurons survive Development Progresses More targets (more neurons) September 15, Target innervation determines which neurons survive Development Progresses More targets (more neurons) September 15, 2010 Lecture V. Cell Birth & Death Fewer targets (fewer neurons) 13

Mouse tumor (sarcoma) transplanted next to developing chick spinal cord causes axon sprouting consistent Mouse tumor (sarcoma) transplanted next to developing chick spinal cord causes axon sprouting consistent with a diffusible factor - a nerve growth factor Levi-Montalcini, R. , & Hamburger, V. (1951) September 15, 2010 Lecture V. Cell Birth & Death 14

A quantitative functional assay for Nerve Growth Factor (NGF) activity, using explanted cultures of A quantitative functional assay for Nerve Growth Factor (NGF) activity, using explanted cultures of sensory ganglia Levi-Montalcini, et al. (1954); Cohen, S. , et al. (1954) Viktor Hamburger Rita Levi-Montalcini Stanley Cohen Hollyday, M. (2001); Abbott, A. (2009). Cohen, S. (1987) September 15, 2010 Lecture V. Cell Birth & Death 15

NGF is the founding member of a large gene family of Neurotrophins (NTs), distantly NGF is the founding member of a large gene family of Neurotrophins (NTs), distantly related to insulin NGF binds as a dimer to its receptor Wiesmann, C. , et al. (1999) September 15, 2010 Lecture V. Cell Birth & Death 16

NGF/Neurotrophins Signal through Trk (tyrosine kinase) Receptors NGF/NT Trk Receptors (Trk. A, Trk. B, NGF/Neurotrophins Signal through Trk (tyrosine kinase) Receptors NGF/NT Trk Receptors (Trk. A, Trk. B, Trk. C, p 75) Multiple Signaling Pathways Via kinases and scaffolding proteins (PIK 3/AKT kinase) Apoptosis pathway (PLC/PKC kinase) (Ras/MAP kinase) Intracellular Ca+2 release, modulation of ion channels September 15, 2010 Lecture V. Cell Birth & Death Gene Activation/ Repression 17

C. elegans is the model organism for molecular genetic studies Bodywall Muscle developmental time C. elegans is the model organism for molecular genetic studies Bodywall Muscle developmental time Hypoderm Sydney Brenner John Sulston Neurons Cuticular Cells Pharynx Intestine Vulva Gonad Germ Cells Muscle Neuronal Cell Death Lineages Brenner, S. (1973) ; Sulston, J. E. , & Horvitz, H. R. (1977); Sulston, J. E. , et al. (1983); White, J. G. , et al. (1982) September 15, 2010 Lecture V. Cell Birth & Death H. Robert Horvitz 18

Programmed Cell Death of single identified neurons can be followed in live worms P Programmed Cell Death of single identified neurons can be followed in live worms P 11 aap September 15, 2010 Lecture V. Cell Birth & Death Sulston, J. E. , & Horvitz, H. R. (1977) 19

2 Classes of C. elegans Cell Death Mutants (pro-survival genes + pro-apoptosis genes) WT 2 Classes of C. elegans Cell Death Mutants (pro-survival genes + pro-apoptosis genes) WT (normal number of cells) (pro-survival genes + pro-apoptosis genes) Mutant class I (fewer cells) (pro-survival genes + pro-apoptosis genes) Mutant class II (extra cells) September 15, 2010 Lecture V. Cell Birth & Death 20

Genetic analysis of cell death genes in C. elegans defines a genetic pathway X Genetic analysis of cell death genes in C. elegans defines a genetic pathway X ced-3(lf) X ced-4(lf) ced-9(lf) X ced-9(lf) excessive cell death (fewer cells) reduced cell death (extra cells) X ced-3(lf) ced-4(lf) animals die as embryos viable reduced cell death (extra cells) viable ced-9 (pro-survival) gene ced-4 ced-3 Cell Death (pro-apoptosis) genes Ellis, R. E. , et al. , (1991); Ellis, R. E. , & Horvitz, H. R. (1991) September 15, 2010 Lecture V. Cell Birth & Death 21

t(14; 18) Chromosomal Translocation Causes Human B-Cell Leukemia by Overexpression of Bcl-2 Chromosome 18 t(14; 18) Chromosomal Translocation Causes Human B-Cell Leukemia by Overexpression of Bcl-2 Chromosome 18 Ig Heavy Chain Chromosome 14 Bcl-2 Chromosome 18 Stanley Korsmeyer Ig Heavy Chain Chromosome 14 Bcl-2 t(14; 18) Chromosomal Translocation September 15, 2010 Lecture V. Cell Birth & Death Cleary, M. L. , et al. , (1986); Haldar, S. , et al. (1990); Korsmeyer, S. J. (1992); Vaux, D. L. , et al. (1988) 22

The “core” Cell Death genes found in C. elegans are conserved as multigene families The “core” Cell Death genes found in C. elegans are conserved as multigene families in vertebrates ced-9 / Bcl-2: ced-4 / Apaf: Bcl-2: B-Cell Leukemia. • “Pro-survival” protein. • Inhibits release of cytochrome C from mitochondria (vertebrates). • Sequesters CED-4 from cytoplasm (worms). Xue, D. , & Horvitz, H. R. (1997) Apaf: Apoptosis activity factor. • “Adaptor” or “scaffold” protein. • Aggregates inactive procaspase, causing auto-activation by proximity. • Requires cytochrome C, and ATP for multimerization (vertebrates). Yang, X. , (1998) ced-3 / Caspase: Cysteine active-site, aspartate cleavage-site, Protease. • “Terminator” protein. • Protease activity when activated by proteolysis. Xue, D. , et al. (1996); Yuan, J. Y. , & Horvitz, H. R. (1990) September 15, 2010 Lecture V. Cell Birth & Death 23

Molecular Model for Apoptosis mitochondria Bcl-2 (ced-9) - single BH 3 domain protein Apaf Molecular Model for Apoptosis mitochondria Bcl-2 (ced-9) - single BH 3 domain protein Apaf (egl-1) (ced-4) caspase (ced-3) (procaspase recruitment) - - - (BH 3 domains) (*Catalysis of the removal of auto-inhibitory caspase domain*) Inactive Procaspase recruited September 15, 2010 Cytochrome C Apaf aggregation activated caspase (cascade) Lecture V. Cell Birth & Death 24

NGF is only one of multiple pathways to the “core” death mechanism, through many NGF is only one of multiple pathways to the “core” death mechanism, through many single-BH 3 proteins Initiation of apoptosis by extracellular ligands (FAS, TNF) NGF “Initiator” caspase-8 Single BH 3 domain protein * * BCL-2 * Single BH 3 domain protein “Core” apoptotic components Gross, A. , et al. , (1999) September 15, 2010 Lecture V. Cell Birth & Death 25

Apaf/Cytochrome C Aggregate into a 7 -Spoke Apoptosome Complex (“Wheel of Death”) Single-particle Electron Apaf/Cytochrome C Aggregate into a 7 -Spoke Apoptosome Complex (“Wheel of Death”) Single-particle Electron Microscope Analysis WD-40 Apaf Cytochrome C +procaspase-9 (x 7? ) CARD (caspase activation and recruitment domain) Apaf gene procaspase-9 Acehan, D. , et al. (2002) September 15, 2010 Lecture V. Cell Birth & Death 26

Single-BH 3 domain molecules integrate multiple signals that trigger apoptosis. Mitochondria integrate “Pro-survival” and Single-BH 3 domain molecules integrate multiple signals that trigger apoptosis. Mitochondria integrate “Pro-survival” and “Pro-death”signals from a family of Bcl-2 -like genes. Pro-survival Pro-death (BH 3) BH 3 Diptheria Toxin (pore forming) p. A September 15, 2010 Bcl-x. L (Bcl-2 like) (BH 3) “Pro-death” Single-BH 3 domain proteins complex with Bcl-2 to release cytochrome C from mitochondria through “giant” mitochondrial ionic channels. Lecture V. Cell Birth & Death Schlesinger, P. H. , et al. , (2002), Fesik, (2000) 27

Molecular Animation of Cell Death Mediated by the FAS pathway Berry, D. (2006) http: Molecular Animation of Cell Death Mediated by the FAS pathway Berry, D. (2006) http: //www. wehi. edu. au/education/wehi-tv/? page=2. September 15, 2010 Lecture V. Cell Birth & Death 28

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What this Lecture was about • Programmed cell death (apoptosis) is a physiological mechanism What this Lecture was about • Programmed cell death (apoptosis) is a physiological mechanism distinct from necrotic cell death. • Apoptosis occurs widely during normal development of the nervous system. • Isolation of specific molecules involved in promoting growth and survival – “trophism, ” e. g. , Nerve Growth Factor (NGF). • What is the “death mechanism” that NGF (and other neruotrophins) inhibit? • Broader implications: controlled cell death in neuroembryology vs uncontrolled cell growth of cancer. • Gene homologies between organisms - humans and worms (nematodes) • Molecular models for apoptosis • How do trophic factors connect to this cell death pathway(s)? September 15, 2010 Lecture V. Cell Birth & Death 30

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