DEFINITION

1. Pathway of cell death induced by a tightly

regulated intracellular program in which
cells destined to die, activate enzymes that
degrade the cells own nuclear DNA & nuclear
& cytoplasmic proteins.

2. Internally programmed cell death.

3. A form of cell death, designed to eliminate

unwanted host cells through activation of
coordinated, internally programmed series of
events effected by a dedicated set of gene
products”.
GENERAL CONSIDERATION
 Apoptotic bodies - Apoptotic cells break up into
fragments, which contain portions of the cytoplasm
and nucleus.

 The plasma membrane of the apoptotic cell and bodies

remains intact,

 Tasty targets for phagocytes.

 The dead cell and its fragments are rapidly devoured,

before the contents have leaked out.

 No leakage
 No inflammatory reaction in the host.
 CAUSES/SIGNIFICANCE

PHYSIOLOGICAL
PATHOLOGICAL
APOPTOSIS IN PHYSIOLOGIC SITUATIONS

 Death by apoptosis is a normal phenomenon

(Suicide)

 It serves to eliminate cells that are no longer

needed,

 To maintain a steady number of various cell

populations in tissues.
PHYSIOLOGICAL SITUATIONS:

 Embryogenesis –Implantation, organogenesis,

developmental involution, and metamorphosis.

 Hormone dependent involution in adults.

For e.g.
1) Endometrial cell breakdown during menstrual cycle,
2) Ovarian follicular atresia in menopause,
3) Regression of the lactating breast after weaning
4) Prostatic atrophy after castration

 Cell death by cytotoxic T cells.

 Elimination of harmful self reactive lymphocytes.

 Cell deletion in proliferating populations.
1) Immature lymphocytes in the bone marrow and thymus
2) B lymphocytes in germinal centers that fail to express
useful antigen receptors
3) Epithelial cells in intestinal crypts.

 Death of host cells that have served their useful

purpose. For e.g.
1) Neutrophils in an acute inflammatory response,
2) Lymphocytes at the end of an immune response
APOPTOSIS IN PATHOLOGIC CONDITIONS

 Apoptosis eliminates cells that are injured

beyond repair without eliciting a host reaction,
thus limiting collateral tissue damage.

 Death by apoptosis is responsible for loss of cells

in a variety of pathologic states
PATHOLOGICAL CONDITIONS
 DNA damage – For e.g. Radiation, cytotoxic anticancer drugs,
and hypoxia can damage DNA

 Accumulation of misfolded proteins – several degenerative

diseases of the central nervous system and other organs.

 Cell death in certain infections –

Viral infections – adenovirus, HIV infections, viral hepatitis

 Cell death in tumours

 Pathological atrophy in parenchymal organs after duct

obstruction - pancreas, parotid gland, and kidney.
HISTOLOGICAL EXAMINATION
 H&E STAIN

 Apoptotic cell appears as an round/oval mass of

intensely eosinophilic cytoplasm with dense
nuclear chromatin fragments.
APOPTOTIC BODIES: CASE OF VIRAL HEPATITIS.
Morphologic features of apoptosis.
A, Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains
brightly eosinophilic cytoplasm and a condensed nucleus.
B, This electron micrography - Some nuclei with peripheral crescents of compacted chromatin, and
others that are uniformly dense or fragmented.
C, Phase contrast micrograph-
left panel, - show blebbing and formation of apoptotic bodies ,
middle panel - a stain for DNA showing nuclear fragmentation
right panel - activation of caspase-3 (, immunofluorescence stain with an antibody specific for the active
form of caspase-3, revealed as red color).
MECHANISM OF APOPTOSIS

 Apoptosis results from the activation of enzymes

called caspases

 Caspases are cysteine proteases that cleave proteins

after aspartic residues.

 Caspases exist as inactive proenzymes, or zymogens,

 Must undergo enzymatic cleavage to become active.

 Active caspases is a marker for cells undergoing

apoptosis
 Initiation phase, - caspases become catalytically active
(depends on balance between production of pro-apoptotic and
anti-apoptotic proteins)

 Two distinct pathways :

A) Intrinsic (mitochondrial pathway)
B) Extrinsic (death receptor pathway)

 Execution phase, during which other caspases trigger the

degradation of critical cellular components.
INTRINSIC (MITOCHNDRIAL) PATHWAY
 Major mechanism of apoptosis.

 Increased permeability of the mitochondrial outer

membrane  Release of death-inducing (pro-apoptotic)
molecule - Cytochrome C into the cytoplasm  initiate
the suicide program of apoptosis.

 Cytochrome C are present in mitchondria that are essential

for life,

 This process is tightly controlled by the BCL2 family of

proteins
BCL2 FAMILY OF PROTEINS
Anti-apoptotic. BCL2, BCL-XL, and MCL1
 they possess four BH domains (called BH1-4).

Pro-apoptotic. BAX and BAK

 they also have four BH domains

Sensors. BAD, BIM, BID, Puma, and Noxa,

 contain only one BH domain, 3rd of 4 BH domains
(called BH3-only proteins).
 Act as sensors of cellular stress and damage
 Regulate the balance between the other two groups,
 Arbiters of apoptosis.
 Cytochrome C released into the cytosol,

 Cytochrome C binds to APAF-1 (apoptosis-activating
factor-1),

 Formation of apoptosome. (wheel-like hexamer)

 This complex bind to caspase-9 and the enzyme cleaves
adjacent caspase-9 molecules.

 Cleavage activates caspase-9

 Thereby activating other pro-caspases

 Active enzymes mediate the execution phase of apoptosis
 THE EXTRINSIC (DEATH RECEPTOR-INITIATED)
PATHWAY

 Outside cell

 Initiated by engagement of plasma membrane death receptors

on a variety of cells.

 Death receptors are members of the TNF receptor family that

contain a cytoplasmic domain

 Domains involved in protein-protein interactions that is called the

death domain because it is essential for delivering apoptotic
signals

 Death receptors - Type 1 TNF receptor (TNFR1) and Fas(CD95)

 The ligand for Fas is called Fas ligand (FasL).

 FasL is expressed on
1) T cells that recognize self antigens (to eliminate self-
reactive lymphocytes),
2) Some cytotoxic T lymphocytes (which kill virus-infected
and tumor cells).

 FasL binds to Fas - >3 molecules of Fas are brought

together  activation of cytoplasmic death domains -
FADD (Fas-associated death domain) binds an inactive
caspase-8 / caspase-10 Multiple pro-caspase-8 cleave one
another  active caspase-8.
 Executioner caspases, such as caspase 3 and 6,
act on many cellular components.

 These caspases, once activated, cleave an

inhibitor of a cytoplasmic DNase and thus make
the DNase enzymatically active; this enzyme
induces cleavage of DNA.

 Caspases also degrade structural components of

the nuclear matrix and thus promote
fragmentation of nuclei.
PROTEIN CROSS-LINKING

Apoptotic bodies
Via transglutaminase activation
-converts soluble cytoplasmic
proteins into covalently
condensed shell leading to
formation of apoptotic bodies.
4. PHAGOCYTIC RECOGNITION

 Apoptotic cells express

phosphatidyl serine
(PS) &
thrombospondin an
adhesive glycoprotein

 Proteins secreted by
phagocytes bind to
apoptotic cells &
opsonize the cells for
phagocytosis.
SUMMARY
NECROSIS VS APOPTOSIS
CLINICOPATHOLOGIC CORRELATIONS:
APOPTOSIS IN HEALTH
AND DISEASE
1) GROWTH FACTOR DEPRIVATION
 Hormone-sensitive cells deprived of the relevant
hormone,

 Unstimulated lymphocytes and neurons deprived

of nerve growth factor die by apoptosis.

 Apoptosis is triggered by the intrinsic (mitochondrial)

pathway

 Decreased synthesis of BCL2 and BCL-XL

 Activation of BIM and other pro-apoptotic members of

the BCL2 family.
2) DNA DAMAGE
 Exposure of cells to radiation or chemotherapeutic
agents induces apoptosis

Mechanism -
 DNA Damaged  p53 protein accumulation in cells  arrests
the cell cycle (at the G1 phase)  to allow time for repair

 If not repaired successfully, p53 triggers apoptosis.

 When TP53 is mutated or absent  No Apoptosis  Cancer

 P53 stimulate several pro-apoptotic members of the BCL2

family, notably BAX, BAK and some BH3-only proteins
3 ) PROTEIN MISFOLDING

 Chaperones in the ER control the proper folding of newly

synthesized proteins,

 Misfolded polypeptides are ubiquitinated and targeted for

proteolysis in proteasomes.

 Inherited mutations or stresses  accumulation of

unfolded or misfolded proteins in ER

 Cellular responses collectively called the unfolded protein

response.
ER stress
 The unfolded protein response  activates signaling
pathways  increase the production of chaperones 
proteasomal degradation of abnormal proteins and slow
protein translation,

 Thus reducing the load of misfolded proteins in the cell

 If this cytoprotective response is unable to cope with the

accumulation of misfolded proteins, the cell activates
caspases and induces apoptosis.

 This process is called ER stress.

 Intracellular accumulation of abnormally folded proteins
causes number of neurodegenerative diseases.

1) Alzheimer
2) Huntington
3) Parkinson diseases
4) Type 2 diabetes.
5) Deprivation of glucose and oxygen, and stress such as
heat, also result in protein
4) APOPTOSIS INDUCED BY THE TNF
RECEPTOR FAMILY
 FasL on T cells binds to Fas on the same or
neighboring lymphocytes.

 This interaction plays a role in the elimination of

lymphocytes that recognize self antigens, and

 Mutations affecting Fas or FasL result in

autoimmune diseases in humans and mice
5) CYTOTOXIC T LYMPHOCYTE-
MEDIATED APOPTOSIS
 Cytotoxic T lymphocytes (CTLs) recognize foreign
antigens presented on the surface of infected host
cells.

 Upon activation, CTLs secrete perforin, a

transmembrane pore-forming molecule, which
promotes entry of the CTL granule serine proteases
called granzymes.

 Granzymes cleave proteins at aspartate residues and

thus activate a variety of cellular caspases.

 CTL kills target cells by directly inducing the

effector phase of apoptosis.
DISORDERS ASSOCIATED WITH DYSREGULATED
APOPTOSIS
CONCLUSION
NECROPTOSIS
Necrosis + Apoptosis = Necroptosis
NECROPTOSIS
 Form of cell death is a hybrid that shares aspects of both
necrosis and apoptosis.

 Morphologically & biochemically -Necrosis,

 Characterized by - loss of ATP, swelling of the cell and
organelles, generation of ROS, release of lysosomal
enzymes and ultimately rupture of the plasma membrane.

 Mechanistically – Apoptosis
 It is triggered by genetically programmed signal
transduction events that culminate in cell death.
 In this respect it resembles programmed cell death, which
is considered the hallmark of apoptosis.
 Necroptosis is called programmed necrosis
 To distinguish it from necrosis driven passively
by toxic or anoxic injury to the cell.

 In sharp contrast to apoptosis, necroptosis does

not result in caspase activation and hence it is
also sometimes

 “Caspase-independent” programmed cell

death.
MECHANISM
 Starts similar to that of the extrinsic form of
apoptosis,

 Ligation of TNFR1 is the most widely studied model

of necroptosis,

 Other signals - ligation of Fas, and sensors of viral

DNA and RNA, genotoxic agents, can also trigger
necroptosis.

 Since TNF can cause both apoptosis and necroptosis,

 Necroptosis involves two unique kinases called

receptor associated kinase 1 and 3 (RIP1 and
RIP3).
 Necroptosis - Physiologic and Pathologic conditions.

 For example,
1) During the formation of the mammalian bone
growth plate
2) Steatohepatitis,
3) Acute pancreatitis,
4) Reperfusion injury,
5) Neurodegenerative diseases such as Parkinson
disease.
6) As a backup mechanism in host defense against
cytomegalovirus etc..
PYROPTOSIS
PYROPTOSIS
 Form of programmed cell death accompanied by the release
of fever inducing Cytokine IL-1

 Biochemical similarities with apoptosis.

 Microbial products that enter the cytoplasm of infected

cells are recognized by cytoplasmic innate immune
receptors and can activate the multiprotein complex called
the inflammasome.

 The function of the inflammasome is to activate caspase- 1,

(also known as interleukin-1β converting enzyme)
which cleaves a precursor form of IL-1 and releases its
biologically active form.
 IL-1 is a mediator of many aspects of inflammation,
including leukocyte recruitment and fever.

 Caspase-1 and Caspase-11 also induce death of the cells.

 Unlike classical apoptosis - Swelling of cells, loss of plasma

membrane integrity, and release of inflammatory
mediators.

 Pyroptosis results in the death of some microbes that gain

access to the cytosol and promotes the release of
inflammasome-generated IL-1.
KEY CONCEPTS
NECROPTOSIS AND PYROPTOSIS
 Necroptosis resembles necrosis morphologically and apoptosis
mechanistically as a form of programmed cell death.

 Necroptosis is triggered by ligation of TNFR1, and viral proteins of

RNA and DNA viruses.

 Necroptosis is caspase-independent but dependent on signaling by the

RIP1 and RIP3 complex.

 Release of cellular contents evokes an inflammatory reaction as in

necrosis.

 Pyroptosis occurs in cells infected by microbes.It involves activation of

caspase-1 which cleaves the precursor form of IL-1 to generate
biologically active IL-1.
 Caspase-1 along with closely related caspase-11 also cause death of
the infected cell.
THANQ