types of cell death
DESCRIPTION
Apoptosis (Programmed Cell Death) :. Necrosis :. Not Self-Initiated Not Stereotypic Can Be Slow “Messy” (injury can spread). Cell-Autonomous Stereotypic Rapid “Clean” (dead cells eaten). Pruitt-Igoe (1972) Saint Louis, MO. Types of Cell Death. Questions : - PowerPoint PPT PresentationTRANSCRIPT
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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
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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
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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.
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Experimental removal of neuronaltarget cells, results in excessive lossof projecting neurons.
Deprived Control
(after Hamburger, 1958, 1977)
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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)
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Loss of neurons timed with innervationof target muscles. Is there a relationship?
IncreasingDevelopmental
TimeTarget
Innervation
Cell Loss
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Number of target cells determines the number of innervation neurons that survive.
Extra target cells(more neurons)
Fewer target cells(fewer neurons)
IncreasingDevelopmental
Time
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Mouse sarcoma transplanted next todeveloping chick nerve cord causes
extra sprouting of neurons. (Diffusible factor suggested)
(Levi-Montalcini and Hamburger, 1951)
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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
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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
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NGF/Neurotrophins Signal Through Trk(tyrosine kinase) Receptors.
NGF/NT
Trk Receptor
Apoptosispathway?
TranscriptionFactor?
Kinases?
Gene Activation/Repression
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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
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(Sulton and Horvitz, 1977)
Programmed Cell Death of single identified cellscan be observed in live worms.
P11aap
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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)
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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
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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
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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
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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)
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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
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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
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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