Types of Cell Death Questions : 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) 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 Pruitt-Igoe (1972) Saint Louis, MO Lecture to Cover: Experimental removal of neuronal target cells, results in excessive loss of projecting neurons. 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. Deprived Control (after Hamburger, 1958, 1977) 1
Massive loss of neurons in embryos occurs during normal development. Loss of neurons timed with innervation of target muscles. Is there a relationship? Lateral Motor Column (40% Loss) Ciliary Ganglion (54% Loss) Increasing Developmental Time Target Innervation Trochlear Nucleus (57% Loss) Cell Loss (after Hamburger, 1975; Landmesser and Pilar, 1974; Cowan and Wenger, 1967) Number of target cells determines the number of innervation neurons that survive. Mouse sarcoma transplanted next to developing chick nerve cord causes extra sprouting of neurons. (Diffusible factor suggested) Increasing Developmental Time (Levi-Montalcini and Hamburger, 1951) Extra target cells (more neurons) Fewer target cells (fewer neurons) 2
Development of a quantitative functional assay for nerve growth factor (NGF) activity, using explanted cultures of sensory ganglia. NGF is the founding member of a large gene family of neurotrophic proteins, distantly related to insulin. mouse NGF M L C L K P V K L G S L E V G H G Q H G G V L A C G R A V Q G A G W - H A G P K L T S V S G P N K G F A K D A A F Y T G human NT-3 M S I L F Y V I F - - - - - - - - - - - - - L A Y L R G I Q G N - - - - - - - N M D Q R S L P E D S L N - - - - - - - - human NT-3/4 M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P L P S C S L P - - - - - - - - human GDNF M K - L W D V V A V C L V L L H T A S A F P L P - - - - - - - - - - - - A G K R P P E A P A E D R S L G R R R A P F A L pig CNTF M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A F A E H S P L T P H chick BDNF M T I L F L T M V - - - - - - - - - - - - - I S Y F S C M K A A - - - - - - - P M K E A S V R G H G - - - - - - - - - - human FGF-5 M S L S F L L L L - - - - - - - - - - - - - - - F F S H L I L S A W A H G E K R L A P K G Q P G P A A T D R N P I G S S rabbit insulin M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A S L A A L L P L mouse NGF R S E V H S V M S M L F Y T L I T A F L I G V Q A E P Y T D S N V P E G D S V P E A H W T K L Q H S L D T A L R R A R S human NT-3 - - - - - - - - S L I I K L I Q A D I L K N K L S K Q M V D V K E N Y Q S T L P K A E - - - - - - - - - A P R E P E R G human NT-3/4 - - - - - - - - I L L L F L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P S V P I E S human GDNF S S D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S N M P E D Y P D Q F D D V M D - - - - - - - - pig CNTF R R D L C S R S I W L A R K I R S D - - L T A L M E A Y V K - - - - - - - - - - - - - - - - - H Q G L N E N I N - - - L chick BDNF - - - - - - - - S L A Y P G L R T H G T L E S L T G P N A G S R G - - L T S L A D T F - - - - - - - - - E H V I E E L L human FGF-5 S R Q S S S - - - - - - - - - - S A M S S S S A S S S P A A S L G S Q G S G L E Q S - - - S F Q W S P S G R R T G S L Y rabbit insulin - - - - - - - - - - L A L L V L C R - - L D P - A Q A F V N - - - - - - - - - - - - - - - - - Q H - - - - - - - - - - - mouse NGF A P T A P I A A R V T G Q T R N I - - T V D P R L F K K - R R L H S P R V L F S T Q P P P T S S D T L D L D - - - - F Q human NT-3 G P A K S A F Q P V I A M D T E L - - - - - - - L R Q Q - R R Y N S P R V L L S D S T P L E P P P L Y L M E D - - - Y V human NT-3/4 Q P P P S T L P P F L A P E W D L - - - - - - - L - - - - - - - - S P R V V L S R G A P A G P P L L F L L E A - G A F R human GDNF - - - - - - - - - - - - - - - - - - - F I Q A T I K R L K R S P D K Q M A V L P R R E R - - - - - - - - - - N R Q A A A pig CNTF D S V D G V P M A S T D R W S E L - - T E A E R L Q E N L R A Y R T F H V M L A R - - - - - - - - - L L E D Q R E H F T chick BDNF D E D Q D I Q P S E E N K D A D L - - - - - - - - - - - - - - Y - T S R V M L S S Q V P L E P P L L F L L E E Y K N Y L human FGF-5 C R V G I G F H L Q I Y P D G K V N G S H E A N M L S V L E I F A V S Q G I V G I R G V F S N K F L A M S K K G K L H A rabbit insulin - - - - - - - - - - - - - - - - L - - C - G S H L V E A L - - Y - - - - - - - - - - - - - - - - - - L V C G E R G F F - (-NGF) (+NGF) (Levi-Montalcini, Hamburger and Cohen, 1954) mouse NGF A H G T I P F N R T H R S K R S S T H P V F H M G E F S V C D S V S V W V - - G D K T T A T D I K G K E V T V L A E V N human NT-3 G S P V V A N R T S R R K - R Y A E H K S - H R G E Y S V C D S E S L W V T - - D K S S A I D I R G H Q V T V L G E I K human NT-3/4 E S A G A P A N R S R R G V S E T A P A S - R R G E L A V C D A V S G W V T - - D R R T A V D L R G R E V E V L G E V P human GDNF A N P E N S R G K G R R G - - - - - - - - - Q R G K N R G C V L T A I H L N V T D L G L G Y E T K E E L I F R Y C S G S pig CNTF P A E D D F H Q A I H T I V L Q V A A F A Y Q L E E L M - - - - - - - - V L L E H K V P P S E A D G T P L S V - - - - - chick BDNF D A A N M S M R V R R H S - - - - - D P A - R R G E L S V C D S T S E W V T A A E K K T A V D M S G A T V T V L E K V P human FGF-5 S A K F T D D C K F R E R F Q E N S Y N T Y A S A I H R T E K T G R E W Y V A L N K R G K A K R G C S - - - - - - - - P rabbit insulin - - - - - Y T P K S R R - - - - - - - - - - E V E E L - - - - - - - - - - - - - - Q V G Q A E L G G G P - - - - - - - - mouse NGF I N - N S V F R Q Y F F E T K C R A S N P V E S G - - - - - - - C R G I D S K H W N S Y C T T T H T F V K A L T T D E K human NT-3 T - G N S P V K Q Y F Y E T R C K E A R P V K N G - - - - - - - C R G I D D K H W N S Q C K T S Q T Y V R A L T S E N N human NT-3/4 A A G G S P L R Q Y F F E T R C K A D N A E E G G P G A G G G G C R G V D R R H W V S E C K A K Q S Y V R A L T A D A Q human GDNF C D A A E T T Y D K I L K N L S R N R R L V S D K V G - - Q A C C R P I - - - - - - - - - - - - - A F D D D L S F L D D pig CNTF - - G G G G L - - - - F E K K L W G L K V L Q - - - - - - - - - - - - - E L S Q W T V R S - - - - - - I R D L R V I S S chick BDNF V P - K G Q L K Q Y F Y E T K C N P K - - - - - - - G Y T K E G C R G I D K R H W N S Q C R T T Q S Y V R A L T M D N K human FGF-5 R V K P Q H I S T H F L P - - - R F K Q S E Q P E L S F T V T V - - - P E K K N P P S P I K S K I P L S A P R K N T N S rabbit insulin - - G A G G L - - - - Q P S A L - - - - - - - - - - - - - - - - - - - - E L A - - - - - - - - - - - - - - - L Q K R G I Rita Levi-Montalcini Viktor Hamburger Stanley Cohen NGF/Neurotrophins Signal Through Trk (tyrosine kinase) Receptors. C. elegans is a great model organism for molecular genetic studies of Cell Death. NGF/NT Bodywall Muscle Hypoderm Kinases? Trk Receptor Apoptosis pathway? developmental time Transcription Factor? 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 ) Gene Activation/ Repression Sydney Brenner John Sulston H. Robert Horvitz 3
Programmed Cell Death of single identified cells can be observed in live worms. 2 Classes of C. elegans Cell Death Mutants WT (pro-survival genes + pro-apoptosis genes) (normal number of cells) Mutant class I (pro-survival genes + pro-apoptosis genes) (fewer cells) Mutant class II (pro-survival genes + pro-apoptosis genes) P11aap (Sulton and Horvitz, 1977) (extra cells) 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 ced-9(lf), ced-3(lf) reduced cell death (extra cells) viable ced-9(lf), ced-4(lf) reduced cell death (extra cells) viable t(14;18) Chromosomal Translocation Causes Human B-cell Leukemia by Overexpression of. Chromosome 18 Ig Heavy Chain Chromosome 14 Chromosome 18 ced-9 (pro-survival) gene ced-4 ced-3 (pro-apoptosis) genes Cell Death Ig Heavy Chain Chromosome 14 t(14;18) Chromosomal Translocation (from Korsmeyer, et al.; Croce, et al.; Sklar, et al.; 1985-1990) Stanley Korsmeyer 4
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). (ced-9) - Molecular Model for Apoptosis mitochondria single BH3 Apaf (egl-1) (ced-4) Cytochrome C (BH3 domains) Apaf: Apoptosis Activity Factor. Adaptor or scaffold protein. Aggregates inactive procaspase, causing auto-activation by proximity. Requires cytochrome C, and ATP for multimerization (vertebrates). caspase (ced-3) (procaspase recruitment) - - (Catalysis of the removal of self-inhibitory caspase domain) Apaf aggregation ced-3 / Caspase: Caspase: Active-site Cysteine, Aspartate Protease. Terminator protein. Protease activity when activated by proteolysis. Recruitment of inactive procaspase activated caspase (cascade) Death NGF maybe one of multiple pathways to the core death mechanism, mediated by many single-bh3 proteins. Apaf/Cytochrome C Aggregate into a 7-Spoke Apoptosome Complex WD-40 Single BH3 Single BH3 WD-40 Cytochrome C Apaf CARD (caspase activation and recruitment domain) Single BH3 Apaf gene +procaspase-9 (x7?) pc-9 (From Gross, McDonnell, and Korsmeyer, 1999) (Acehan, et al., 2002) 5
The C. elegans egl-1 gene resembles vertebrate single BH3 domain molecules that trigger apoptosis. Mitochondria integrate Pro-survival and Pro-death signals from a family of -like genes. Pro-survival Pro-death (BH3) (BH3) Pro-death Single-BH3 s complex with to release cytochrome C from mitochondria. BH3 + BH3 Diptheria Toxin (pore forming) Bcl-xL ( like) BID (single-bh3) (from Fesik, 2000) 6