Types of Cell Death Apoptosis (Programmed Cell Death) : Cell-Autonomous Stereotypic Rapid Clean (dead cells eaten) Necrosis : Not Self-Initiated Not Stereotypic Can Be Slow Messy (injury can spread) Apoptosis from Greek apo meaning separation & ptosis for falling off Pruitt-Igoe (1972) Saint Louis, MO (Kerr JFR, Wyllie AH & Currie AR, 1972) Questions : Is Apoptosis a real biological phenomenon (controlled by a genetic program)? How are cells killed? What turns apoptosis on and off? Can this program be altered for good or evil? i.e. cancer too little apoptosis or neurodegenerative diseases too much apoptosis Lecture to Cover: Classic experiments describing the phenomenology of cell death. Identification of NGF and other protein trophic factors. Cloning of genes comprising the death mechanism. Molecular model for apoptosis. 1
Experimental removal of neuronal target cells, results in excessive loss of projecting neurons Massive loss of neurons in embryos occurs during normal development (!) Lateral Motor Column (40% Loss) Ciliary Ganglion (54% Loss) Trochlear Nucleus (57% Loss) Deprived Control (after Hamburger, 1958, 1977) (Hamburger, 1975; Landmesser and Pilar, 1974; Cowan and Wenger, 1967) Loss of neurons timed with innervation of target muscles. Is there a relationship? Number of target cells determines the number of innervation neurons that survive Motoneurons Target Muscles Increasing Developmental Time Target Innervation Increasing Developmental Time (Not all neurons innervate targets) Cell Loss Extra target cells (more neurons) Fewer target cells (fewer neurons) 2
Mouse sarcoma transplanted next to developing chick nerve cord causes extra sprouting of neurons. (Diffusible factor suggested) Development of a quantitative functional assay for Nerve Growth Factor (NGF) activity, using explanted cultures of sensory ganglia (-NGF) (+NGF) (Levi-Montalcini, Hamburger and Cohen, 1954) (Levi-Montalcini and Hamburger, 1951) Rita Levi-Montalcini Viktor Hamburger Stanley Cohen NGF is the founding member of a large gene family of Neurotrophins (NTs), distantly related to insulin Mouse NGF NGF/Neurotrophins Signal through Trk (tyrosine kinase) Receptors NGF/NT Trk Receptors (TrkA, TrkB, TrkC, p75) Rabbit insulin NGF binds as a dimer to its receptor Multiple Signaling Pathways via pathwayspecific kinases and scaffolding proteins (PLC/PKC kinase) Intracellular Ca +2 release, modulation of ion channels (PIK3/AKT kinase) Apoptosis pathway (Ras/MAP kinase) Gene Activation/ Repression (Weismann, et al., 1999) 3
C. elegans is a great model organism for molecular genetic studies of Cell Death Programmed Cell Death of single identified neurons can be followed in live worms Bodywall Muscle Hypoderm developmental time Neurons Cuticular Cells Pharynx Intestine Vulva Gonad Germ Cells Muscle Neuronal Cell Death Lineages (Brenner, 1973; Sulston and Horvitz, 1977; White, Horvitz, Sulston, 1982; Sulston, Schierenberg, White, Thomson, 1983 ) P11aap Sydney Brenner John Sulston H. Robert Horvitz (Sulton and Horvitz, 1977) 2 Classes of C. elegans Cell Death Mutants WT (pro-survival genes + pro-apoptosis genes) Genetic analysis of cell death genes in C. elegans defines a genetic pathway ced-9(lf) excessive cell death (fewer cells) animals die as embryos ced-3(lf) reduced cell death (extra cells) viable ced-4(lf) reduced cell death (extra cells) viable (normal number of cells) Mutant class I (pro-survival genes + pro-apoptosis genes) ced-9(lf), ced-3(lf) reduced cell death (extra cells) viable ced-9(lf), ced-4(lf) reduced cell death (extra cells) viable (fewer cells) Mutant class II (pro-survival genes + pro-apoptosis genes) ced-9 (pro-survival) gene ced-4 ced-3 (pro-apoptosis) genes Cell Death (Horvitz lab, mid-1980 to early-1990s) (extra cells) 4
t(14;18) Chromosomal Translocation Causes Human B-Cell Leukemia by Overexpression of Chromosome 18 Ig Heavy Chain Chromosome 14 The core Cell Death genes found in C. elegans are evolutionarily conserved as multigene families in vertebrates ced-9 / : ced-4 / Apaf: : B-Cell Leukemia. Pro-survival protein. Inhibits release of cytochrome C from mitochondria (vertebrates). Sequesters CED-4 from cytoplasm (worms). (Horvitz, Kim Labs) (Vaux, Cory & Adams, 1988) (Korsmeyer, et al.; Croce, et al.; Sklar, et al.; 1985-1990) Stanley Korsmeyer Chromosome 18 Ig Heavy Chain Chromosome 14 t(14;18) Chromosomal Translocation ced-3 / Caspase: Apaf: Apoptosis activity factor. Adaptor or scaffold protein. Aggregates inactive procaspase, causing auto-activation by proximity. Requires cytochrome C, and ATP for multimerization (vertebrates). (Horvitz, Wang, Baltimore Labs) Caspase: Cysteine active-site, aspartate cleavage-site, Protease. Terminator protein. Protease activity when activated by proteolysis. (Horvitz, Yuan Labs) (ced-9) - Molecular Model for Apoptosis mitochondria single BH3 (egl-1) Apaf (ced-4) Cytochrome C (BH3 domains) NGF is only one of multiple pathways to the core death mechanism, mediated by many single-bh3 proteins Initiation of apoptosis by extracellular ligands (FAS, TNF) NGF caspase (ced-3) (procaspase recruitment) - - (Catalysis of the removal of auto-inhibitory caspase domain) Apaf aggregation Initiator caspase-8 Single BH3 Single BH3 BCL-2 Single BH3 Recruitment of inactive procaspase activated caspase (cascade) Death Core apoptotic components (Gross, McDonnell, and Korsmeyer, 1999) 5
Molecular Details: Apaf/Cytochrome C Aggregate into a 7-Spoke Apoptosome Complex ( Wheel of Death ) Single-particle Electron Microscope Analysis Molecular Details: Single-BH3 domain molecules integrate multiple signals that trigger apoptosis. Pro-survival Mitochondria integrate Pro-survival and Pro-death signals from a family of -like genes. Pro-death WD-40 WD-40 Cytochrome C Apaf CARD (caspase activation and recruitment domain) (BH3) (BH3) Pro-death Single-BH3 s complex with to release cytochrome C from mitochondria through giant mitochondrial ionic channels. Apaf gene +procaspase-9 (x7?) procaspase-9 Diptheria Toxin (pore forming) Bcl-xL ( like) BH3 (Fesik, 2000) pa (Acehan, et al., 2002) (Schlesinger and Saito, 2006) Molecular Animation of Cell Death Mediated by the FAS pathway Courtesy of Dr. Drew Berry The Walter and Eliza Hall Institute of Medical Research Melbourne, Australia 6