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

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.

Experimental removal of neuronaltarget cells, results in excessive lossof projecting neurons.

Deprived Control

(after Hamburger, 1958, 1977)

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)

Loss of neurons timed with innervationof target muscles. Is there a relationship?

IncreasingDevelopmental

TimeTarget

Innervation

Cell Loss

Number of target cells determines the number of innervation neurons that survive.

Extra target cells(more neurons)

Fewer target cells(fewer neurons)

IncreasingDevelopmental

Time

Mouse sarcoma transplanted next todeveloping chick nerve cord causes

extra sprouting of neurons. (Diffusible factor suggested)

(Levi-Montalcini and Hamburger, 1951)

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)

RitaLevi-Montalcini

ViktorHamburger

StanleyCohen

NGF is the founding member of a large gene familyof 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 Ghuman 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 Lpig CNTF M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A F A E H S P L T P Hchick 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 Srabbit 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 Shuman 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 Ghuman NT-3/4 - - - - - - - - I L L L F L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P S V P I E Shuman 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 - - - Lchick 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 Lhuman 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 Yrabbit 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 Qhuman 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 Vhuman 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 Rhuman 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 Apig 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 Tchick 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 Lhuman 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 Arabbit insulin - - - - - - - - - - - - - - - - L - - C - G S H L V E A L - - Y - - - - - - - - - - - - - - - - - - L V C G E R G F F -

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 Nhuman 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 Khuman 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 Phuman 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 Spig 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 Phuman 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 - - - - - - - - Prabbit 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 Khuman 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 Nhuman 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 Qhuman 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 Dpig 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 Schick 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 Khuman 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 Srabbit insulin - - G A G G L - - - - Q P S A L - - - - - - - - - - - - - - - - - - - - E L A - - - - - - - - - - - - - - - L Q K R G I

Neurotrophin Gene Family

NGF/Neurotrophins Signal Through Trk(tyrosine kinase) Receptors.

NGF/NT

Trk Receptor

Apoptosispathway?

TranscriptionFactor?

Kinases?

Gene Activation/Repression

C. elegans is a great model organism for molecular genetic studies of Cell Death.

SydneyBrenner

JohnSulston

H. RobertHorvitz

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

deve

lopm

enta

l tim

e

Neuronal Cell Death Lineages

(Sulton and Horvitz, 1977)

Programmed Cell Death of single identified cellscan be observed in live worms.

P11aap

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)

ced-3

ced-4 CellDeath

ced-9(pro-survival)

gene (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 dieas embryos

ced-3(lf) reduced cell death (extra cells) viableced-4(lf) reduced cell death (extra cells) viable

ced-9(lf), ced-3(lf) reduced cell death (extra cells) viableced-9(lf), ced-4(lf) reduced cell death (extra cells) viable

t(14;18) Chromosomal Translocation Causes Human B-cell Leukemia

by Overexpression of Bcl-2.

Bcl-2Chromosome 18

Ig Heavy ChainChromosome 14

Bcl-2Chromosome 18

Ig Heavy ChainChromosome 14

t(14;18) Chromosomal Translocation

Bcl-2

(from Korsmeyer, et al.; Croce, et al.; Sklar, et al.; 1985 - 1990)

Stanley Korsmeyer

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.

CeHu

CeHu

CeHu

Molecular Model for Apoptosis

activatedcaspase

(cascade)

(BH3 domains)

Apaf aggregation

single BH3domain protein

(egl-1)

mitochondria

Bcl-2(ced-9)

Apaf(ced-4)

caspase(ced-3)

---

Death

Recruitment ofinactive

procaspase

(procaspase recruitment)

------

Cytochrome C

(Catalysis of the removal of

self-inhibitory caspase domain)

NGF maybe one of multiple pathways to the “core” death mechanism, mediated

by many single-BH3 proteins.

(From Gross, McDonnell, and Korsmeyer, 1999)

Single BH3domain protein

Single BH3domain protein

Single BH3domain protein

PDGFNGF

The C. elegans egl-1 gene resembles vertebrate single BH3 domain molecules that trigger apoptosis.

“Pro-death” Single-BH3 domain proteins complexwith 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)(BH3)

Pro-deathPro-survival

Diptheria Toxin(pore forming)

+

Bcl-xL(Bcl-2 like)

BID(single-BH3)

BH3BH3

Bcl-2 Bax

Bik

Apaf/Cytochrome C Aggregate into a 7-Spoke Apoptosome Complex

+procaspase-9 (x7?)

pc-9

(Acehan, et al., 2002)

Apaf gene

Cytochrome CCARD(caspase activationand recruitment domain)

ApafWD-40

WD-40