Pathophysiology: apoptosis

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Apoptosis and Diseases
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• Concept
• Apoptotic process and changes
• Key molecules and Major pathways
• Techniques to detect apoptosis
• Apoptosis-related diseases
• Insufficient apoptosis in diseases
• Excessive apoptosis in diseases
• Coexistence of insufficient and excessive
  apoptosis in diseases
• Principles of treatment

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What is Apoptosis ?

• Apoptosis refers to the process in which the
  dying procedures that have been in advance
  deposited in cell are triggered by various causes
  from in vitro and in vivo, and eventually cause
  cell death.
• Programmed cell death(PCD)

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Causes and Process of Apoptosis
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Apoptotic changes ---Morphological changes in
apoptosis ---Biochemical Changes in Apoptosis
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Morphological changes in apoptosis

• Cell membrane
• Cytoplasm
• Cell nucleus
• Apoptotic body
• Phagocytose

Apoptotic Bodies
Changes of Cell membrane
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Apoptosis and Necrosis
Apoptosis Necrosis
Nature Physiological or pathological specific Pathological, accidental
Stimulus Mild Strong
Biochemistry Active, energy-dependent, new protein synthesis Passive, energy-independent, no protein synthesis
DNA Specific degradation, ladder (180-200 bp) Random degradation
Morphology Intact, shrinkage, condensation Lysis, swelling
Inflammation No Yes
Apoptotic body Yes No
Gene regulation Yes No
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Morphological differences in apoptosis and
necrosis
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Biochemical Changes in Apoptosis

• Caspase activation
• Endonuclease activation

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Caspases (cysteine-containing
aspartate-specific proteases)

• Most apoptotic proteolytic cleavage results from
  the action of caspases
• Caspases are activated by proteolytic cleavage
• Removal of prodomain and linker region
• Assembly of the large and small subunits into an
  active enzyme complex
• Two heterodimers interacting via the small
  subunits to form a tetramer with two catalytic
  sites
• Family membersgt14

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Caspase functions and structure
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Classification of Caspases
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Caspase-deficient mice Knockout
Phenotype Caspase-1 Viable impaired processing
of IL-1 resistant to endotoxic shock. Caspase-2
Viable excess numbers of female germ cells
oocytes resistant to
chemotherapeutic drugs B lymphoblasts resistant
to granzyme B
accelerated death of facial neurons during
development and of
sympathetic neurons deprived of NGF. Caspase-3
Lethality at 35 weeks of age defective neuronal
apoptosis T cells
resistant to antigen-induced death abnormal
apoptotic morphology in
dying cells. Caspase-8 Lethality around E12.5
hyperemia and abnormal heart muscle
development MEFs resistant to TNF, Fas
and DR3 but sensitive to
UV irradiation, etoposide, staurosporine, serum
deprivation. Caspase-9 Perinatal lethal
impaired neuronal apoptosis ES cells, MEFs and
thymocytes generally
resistant to intrinsic death stimuli such as
DNA damage, though
resistance depends on cell type. Caspase-11
Viable impaired processing of caspase-1, IL-1
resistant to endotoxic
shock. Caspase-12 Viable embryonic fibroblasts
are resistant to ER stress.
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• Caspases activation
• Death receptor pathway caspase 8
• Mitochondrial pathway caspase 9
• ER stress pathway caspase 12

(CAD caspase-activated deoxyribonulease)
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• Caspase substrates
• ICADDNA??
• PARP??DNA??
• ??????gelsolin, laminA
• ????????Bcl-2
• etc

Cleavage of death substrates
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Role of Endonucleasedegrade DNA
Signaling activation
Endonuclease
Zn2
Ca2 Mg2
180-200 bp
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Apoptotic substrates
( DNA-PKCS, DNA protein kinase catalytic subunit
HnRNP, heteronuclear ribonucleoproteins
ICAD,inhibitor caspase activated
deoxyribonuclease FAK,focal adhesion kinase
GAS,growth arrest specific gene-2 GDI, GDP
dissociation inhibitor NuMA,nuclear mitotic
apparatus PAK,p21 activated kinasePARP,
poly(ADP-ribose) polymerase cPLA2, cytoplasmic
phospholipase A2 RFC-140, replication factor C
SAF-A,scaffold attachment factor-A U1-70kDa,
U1-specific 70-kDa protein )
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Regulators of ApoptosisBcl2 family proteinsIAP
(Inhibitors of Apoptosis Proteins)
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Bcl2 family killers and protectors

• Two groups (gt15 members) to keep the balance
  between apoptosis and survival
• ---Suppressors of apoptosis Bcl2, BclXL, BclW,
  Bag1, Mcl1, A1, etc
• ---Activators of apoptosis Bax, Bok, Hrk, Bnip3,
  Bim, Bik, BclXs, Bik, Blk, Bid, Bak, Bad, etc.
• Forms heterodimers

• On the cytoplasmic face of the outer
  mitochondrial membrane, endoplasmic reticulum,
  and nuclear envelope
• In hematopoietic cell, epithelial cell,
  lymphocyte, nerve cell, and various cancer cells

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Bcl-2 Regulate the release of pro-apoptotic
molecules from mitochondria Structure of Bcl-2
family
TM transmembrane region BH Bcl-2 homology
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Bax

• Apoptotic stimuli induce translocation of Bax
  from cytosol to mitochondria
• create pores in the outer membrane of
  mitochondria of sufficient size to allow
  cytochrome C to escape

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IAPs---family members c-IAP1,c-IAP2,XIAP,NAIP,s
urvivin---preventing some procaspases
activation, or inhibiting caspase activity.
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• Apoptosis pathways and related genes
• Death Receptor induced apoptosis
• Mitochondria Integrator of Apoptosis
• ER stress pathway
• Others

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Death Factor and Death Receptor Family
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Death receptor induced apoptosis

• Fas ( factor associated suicide)
• Homologous cytoplasmic domain death domain (DD)
• Interacts with each other through DD
• Anti-apoptotic pathway NF-kB pathway
• TNF rarely induces apoptosis unless protein
  synthesis is inhibited
• Decoy receptors

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Death Receptor Signaling
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Apoptosis signaling by CD95, TNFR1, and DR3
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Apoptosis signaling by DR4 and DR5 and its
modulation by decoy receptors
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Three Types of Killing by the Fas and FasL System

1. Activation-induced suicide of T cells
2. CTL-mediated killing of target cells
3. Killing of inflammatory cells in immune privilege
   sites and killing of CTL by tumor cells

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Mitochondria Integrator of Apoptosis
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Current models of the intracellular pathways
leading to trophic factor mediated cell survival
in mammalian cells
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Current models of the intracellular pathways
leading to apoptosis induced by withdrawal of
trophic factor
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ER and Apoptosis
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Cross-talking among Organelles and Molecules in
Apoptosis
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p53 Mediated Apoptosis
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p53-Inducible Apoptosis Related Genes

• Scotin localized to the ER and the nuclear
  membrane
• PERPsimilarity to PMP-22/gas3 tetraspan membrane
  protein
• NOXA A member of Bcl-2 family
• BAX
• KILLERS/DR5
• FAS
• P53AIP1 p53-regulated apoptosis-inducing
  protein 1, leads to apoptosis via dissipation of
  mitochondrial??m
• PIDD A new death domain containing protein
• PIG P53 induced genes,related to ROS production
• IGFBP

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The Mammalian DNA Damage Checkpoint
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Four patterns of death from apoptosis to necrosis

• Apoptosis is observed almost exclusively when
  caspases, in particular caspase-3, are activated.
• Apoptosis-like PCD chromatin condensation less
  compact without other apoptotic features
  caspase-independent apoptosis
• Necrosis-like PCD no chromatin condensation with
  chromatin clustering to speckles. Usually
  involves specialized caspase-independent
  signalling pathways. aborted apoptosis
• Accidental necrosis/cell lysis associated with
  cellular oedema (organelle swelling) and devoid
  of zeiosis

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Techniques to detect apoptosis

• Morphological studies
• DNA ladder
• TUNEL
• Flow cytometry
• Externalization of Phosphatidylserine
• Activation of caspases and cleavage of their
  substrates

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Ultrastructural feathers of Normal and Apoptotic
Cell
Induced Apoptosis of Cultured Rat Hepatocytes
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DNA Ladder Pattern Seen in Diospyrin diethyl
ether Induced Apoptotic Cell
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Fragmented DNA can be labeled by
Terminal deoxynucleotidyl
transferase (TdT)
mediated
deoxyUridine Nucleotide
(dUTP)-End Labeling
(TUNEL)
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Flowcytometric Analysis of Cellular DNA Content
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Externalization of Phosphatidylserine
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Phosphatidylserine on the surface of apoptotic
cellsstained with annexin V (green)
Due to Caspase-3-mediated cleavage and
activation of scramblase PKC activation
Inactivated amino- phospholipid translocase
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Activation of Caspase 3 and Cleavage of Its
Substrates, PARP and D4-GDI