1. 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) Pruitt-Igoe (1972) Saint Louis, MO

2. 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

3. 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.

4. Experimental removal of neuronal target cells, results in excessive loss of projecting neurons. Deprived Control (after Hamburger, 1958, 1977)

5. Massive loss of neurons in embryos occurs during normal development. Lateral Motor Column (40% Loss) Ciliary Ganglion (54% Loss) Trochlear Nucleus (57% Loss) (after Hamburger, 1975; Landmesser and Pilar, 1974; Cowan and Wenger, 1967)

6. Loss of neurons timed with innervation of target muscles. Is there a relationship? Increasing Developmental Time Target Innervation Cell Loss

7. Number of target cells determines the number of innervation neurons that survive. Extra target cells (more neurons) Fewer target cells (fewer neurons) Increasing Developmental Time

8. Mouse sarcoma transplanted next to developing chick nerve cord causes extra sprouting of neurons. (Diffusible factor suggested) (Levi-Montalcini and Hamburger, 1951)

9. 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) Rita Levi-Montalcini Viktor Hamburger Stanley Cohen

10. NGF is the founding member of a large gene family of neurotrophic proteins, distantly related to insulin. Neurotrophin Gene Family

11. NGF/Neurotrophins Signal Through Trk (tyrosine kinase) Receptors. NGF/NT Trk Receptor Apoptosis pathway? Transcription Factor? Kinases? Gene Activation/ Repression

12. C. elegans is a great model organism for molecular genetic studies of Cell Death. Sydney Brenner John Sulston H. Robert Horvitz Hypoderm Bodywall Muscle (Brenner, 1973; Sulston and Horvitz, 1977; White, Horvitz, Sulston, 1982; Sulston, Schierenberg, White, Thomson, 1983 ) Cuticular Cells Neurons Pharynx Intestine Vulva Germ Cells Gonad Muscle developmental time Neuronal Cell Death Lineages

13. (Sulton and Horvitz, 1977) Programmed Cell Death of single identified cells can be observed in live worms. P11aap

14. 2 Classes of C. elegans Cell Death Mutants (pro-survival genes + pro-apoptosis genes) WT (pro-survival genes + pro-apoptosis genes) Mutant class I (pro-survival genes + pro-apoptosis genes) Mutant class II (fewer cells) (extra cells) (normal number of cells)

15. ced-3 ced-4 Cell Death ced-9 (pro-survival) gene (pro-apoptosis) genes Genetic analysis of cell death genes in C. elegans defines a genetic pathway.

16. t(14;18) Chromosomal Translocation Causes Human B-cell Leukemia by Overexpression of Bcl-2. Bcl-2 Chromosome 18 Ig Heavy Chain Chromosome 14 Bcl-2 Chromosome 18 Ig Heavy Chain Chromosome 14 t(14;18) Chromosomal Translocation Bcl-2 (from Korsmeyer, et al.; Croce, et al.; Sklar, et al.; 1985 - 1990) Stanley Korsmeyer

17. The “Core” Cell Death genes found in C. elegans are evolutionarily conserved as multigene families in vertebrates. ced-9 / Bcl-2: Bcl-2: B-cell Leukemia. “Pro-survival” protein. Inhibits release of cytochrome C from mitochondria (vertebrates). Sequesters CED-4 from cytoplasm (worms). ced-4 / Apaf: Apaf: Apoptosis Activity Factor. “Adaptor” or “scaffold” protein. Aggregates inactive procaspase, causing auto-activation by proximity. Requires cytochrome C, and ATP for multimerization (vertebrates). ced-3 / Caspase: Caspase: Active-site Cysteine, Aspartate Protease. “Terminator” protein. Protease activity when activated by proteolysis. Ce Hu Ce Hu Ce Hu

18. Molecular Model for Apoptosis activated caspase (cascade) (BH3 domains) Apaf aggregation single BH3 domain protein (egl-1) mitochondria Bcl-2 (ced-9) Apaf (ced-4) caspase (ced-3) - - - Death Recruitment of inactive procaspase (procaspase recruitment) - - - - - - Cytochrome C (Catalysis of the removal of self-inhibitory caspase domain)

19. NGF maybe one of multiple pathways to the “core” death mechanism, mediated by many single-BH3 proteins. (From Gross, McDonnell, and Korsmeyer, 1999) Single BH3 domain protein Single BH3 domain protein Single BH3 domain protein PDGF NGF

20. The C. elegans egl-1 gene resembles vertebrate single BH3 domain molecules that trigger apoptosis. “Pro-death” Single-BH3 domain proteins complex with Bcl-2 to release cytochrome C from mitochondria. (from Fesik, 2000) Mitochondria integrate “Pro-survival” and “Pro-death” signals from a family of Bcl-2-like genes. (BH3) Pro-deathPro-survival Diptheria Toxin (pore forming) + Bcl-xL (Bcl-2 like) BID (single-BH3) BH3 Bcl-2 Bax Bik

21. Apaf/Cytochrome C Aggregate into a 7-Spoke Apoptosome Complex +procaspase-9 (x7?) pc-9 (Acehan, et al., 2002) Apaf gene Cytochrome C CARD (caspase activation and recruitment domain) Apaf WD-40