This document provides an overview of apoptosis, or programmed cell death. It begins with an introduction defining apoptosis and its importance in development, aging, and disease. It then describes the main types and mechanisms of apoptosis, including the extrinsic and intrinsic pathways. Key features of apoptosis like morphology, assays to detect it, and differences from necrosis are summarized. The document concludes by discussing other forms of programmed cell death and clinical applications of understanding apoptosis.

1. APOPTOSIS
Presentation by -Dr Sindhu, 1st yr resident
Moderator – Dr Sharma sir
Professor
Department of pathology

2. OVERVIEW
INTRODUCTION
TYPES OF APOPTOSIS
MECHANISM OF APOPTOSIS
MORPHOLOGY OF APOPTOSIS
NECROSIS VS APOPTOSIS
ASSAYS OF APOPTOSIS
OTHER TYPES OF PROGRAMMED CELL DEATH

3. INTRODUCTION
What is apoptosis ?
• The term apoptosis was first recognized in 1972 by the distinctive
morphologic appearance of membrane bound fragments derived from cells
and named after the Greek designation for ‘’falling off’’.
• Apoptosis is a type of cell death that is induced by a tightly regulated suicide
program in which cells destined to die activate intrinsic enzymes that
degrade the cells genomic DNA and nuclear and cytoplasmic proteins.
• Apoptosis has since been recognized and accepted as a distinctive and
important mode of “programmed” cell death, which involves the genetically
determined elimination of cells.

4. Why apoptosis ?
• Normal during development and aging – homeostatic
mechanism
• Defense mechanism - immune reactions or when cells are
damaged by disease or noxious agents
• Stimulus- physiological or pathological
 Irradiation
Chemotherapy
Corticosteroids

5. • Does all cells respond to stimulus?
• Cells express Fas or TNF receptors that can lead to apoptosis via
ligand binding and protein cross-linking.
• Cells have a default death pathway that must be blocked by a
survival factor such as a hormone or growth factor
• Apoptosis vs Necrosis, two processes that can occur independently,
sequentially, as well as simultaneously

6. • Apoptosis is a coordinated and often energy-
dependent process that involves the activation of a
group of cysteine proteases called “caspases” and a
complex cascade of events.

7. TYPES OF APOPTOSIS
PHYSIOLOGICAL APOPTOSIS
The role of apoptosis in normal physiology is as
significant as that of its counterpart, mitosis.
PATHOLOGICAL APOPTOSIS
Abnormalities in cell death regulation can be a
significant component of diseases

8. PHYSIOLOGICAL APOPTOSIS
 Developmental processes
 To get rid of the body of pathogen-invaded cells and is a vital component of
wound healing - the removal of inflammatory cells and the evolution of
granulation tissue into scar tissue.
 Dysregulation of apoptosis during wound healing can lead to pathologic forms of
healing such as excessive scarring and fibrosis.
Apoptosis is also needed to eliminate activated or auto-aggressive immune cells
either during maturation.
 Apoptosis is central to remodeling in the adult, such as the follicular atresia of the
postovulatory follicle and post-weaning mammary gland involution.
Age induced apoptosis- oxidate stress

9.  PATHOLOGICAL APOPTOSIS
Damage to host cell DNA e.g. in therapy for cancer by
irradiation or by chemotherapy, mild thermal injury or hypoxia
Accumulation of misfolded proteins formed by free radical
injury or from mutations e.g. in degenerative diseases of CNS
e.g.Alzheimer’s disease, Parkinson’s disease, chronic infective
dementias.
Certain viral infections e.g. councilman bodies in viral hepatitis,
progressive depletion of CD4+T cells in HIV infection.
Cell death by cytotoxic Tcells in transplant rejection reaction.
Pathologic atrophy of organs and tissues on withdrawal of
stimuli e.g. prostatic atrophy after orchiectomy, atrophy of
kidney or salivary gland on obstruction of ureter or ducts
respectively.

10. FACTORS
?
CELL
INJURY
? ? ? ? ?
NECROSIS APOPTOSIS
• INTRACELLULAR FACTORS
• Availability of CASPASES
• ATP
• EXTRACELLULAR FACTORS
• Type of death signal
• Tissue type
• Developmental stage of tissue
• Duration and intensity of
stimulus

11. MECHANISM OF APOPTOSIS

12. EXTRINSIC PATHWAY
• Apoptosis generally induced by two pathways
1. Extrinsic pathway
2. Intrinsic pathway
• All this pathways converge onto common pathway
• These pathways involve the action of the cysteine proteases called
Caspases that cleaves there target proteins after an aspartic acid
residue.

13. • The extrinsic pathway is triggered
by the binding of ligands to the
transmembrane death receptors,
such as tumour necrosis factor
receptor 1 (TNFR1), and Fas
receptor (FasR).
• The ligand binding causes
clustering of the cytoplasmic
domains of these receptors, which,
in turn, recruits adapter proteins
containing corresponding death
domains

14. • The death domains of these
adapter proteins serve as a binding
site for different upstream (initiator)
procaspases (such as caspase-8 and
caspase-10) to form a death-
inducing signaling complex (DISC),
which activates these caspases
• The activated upstream caspases
then activate downstream
(executioner) caspases (such as
caspase-3, caspase-6, and caspase-
7) to execute the cell death.

15. • A prominent feature of apoptosis is the absence of
systemic or localised damage to other cells owing to
lack of inflammation during its initiation, completion,
and the clearance of the dead cells.
• The apoptotic cell achieves this by carefully wrapping
the cell membrane around the new fragmented cell,
forming apoptotic bodies.

16. • Nucleotides such as adenosine 5′-
triphosphate (ATP) and uridine-5′-
triphosphate (UTP), which are
released by the apoptotic cell,
serve as the ‘find-me' signal for
macrophages.
• Once a macrophage gain sufficient
proximity, the apoptotic bodies
enable the former to engulf them
by displaying phosphatidyl serine
(the ‘eat-me signal’) on their
surface

17. INTRINSIC PATHWAY
• The intrinsic pathway, also called the
mitochondrial pathway, involves the release
of cytochrome c from the mitochondria to
the cytoplasm in response to cellular stress.
• The cytochrome c, in association with
apoptotic protease-activating factor-1 (Apaf-
1), forms an apoptosome. Within
apoptosome, procaspase-9 is activated to
become caspase-9. Caspase-9, in turn,
activates downstream effector caspases

18. • The mitochondria further release
proapoptotic proteins such as
apoptosis-inducing factor (AIF) and
endonuclease G.
• AIF translocates to the nucleus and
facilitates DNA fragmentation and
chromatin condensation. After this,
‘stage I’ chromatin condensation (also
called ring condensation) ensues the
translocation of endonuclease G to the
nucleus leading to further
fragmentation of the DNA.
• Finally, the caspase-activated DNase
(CAD) is translocated to the nucleus,
leading to ‘stage II condensation’ (the
necklace condensation)

19. REGULATORS OF INTRINSIC PATHWAY
(MITOCHONDRIA)
• The mitochondrial phase of apoptosis is regulated by the Bcl-2 family of
proteins.
• The proapoptotic proteins of this family, such as BAX and BAK, create pores
on the mitochondrial membrane in response to apoptotic stimuli, facilitating
the release of cytochrome c.
• Meanwhile, other proapoptotic proteins, such as BAD, BIM, BID, Puma, Noxa,
keep the anti-apoptotic protein, Bcl-2, inactive. The released cytochrome c,
through its interaction with Apaf-1, activates caspase-9. Meanwhile, the
mitochondria release a second molecule, aptly named, second mitochondria-
derived activator of caspase (SMAC) that inhibits IAP (for inhibitor of
apoptosis) proteins preventing the latter from deactivating caspase-9.
Caspase-9, thus fully enabled, carries out the cell death by activating

20. MECHANISM OF APOPTOSIS

21. MORPHOLOGY OF APOPTOSIS
• LIGHT MICROSCOPE
I. CELL SHRINKAGE
II. CHROMATIN CONDENSATION
• Aapoptotic cell -round or oval mass
with dark eosinophilic cytoplasm and
dense purple nuclear chromatin
fragments

22. • Apoptotic thymic lymphocytes The
arrow indicates a fragmented
section of a nucleus and the
arrowhead most likely indicates an
apoptotic body that seems to
contain predominantly cytoplasm
Macrophage with engulfed
intracytoplasmic apoptotic bodies.
This macrophage is also called a
“tingible body macrophage.”

23. NECROSIS
• Toxic
• Energy independent
• Uncontrolled
• Affects large group of cells
• cell swelling
• pyknosis, karyorrhexis,
karyolysis
• disrupted cell membrane
• cytoplasm released
• inflammation usually present
• homeostatis/ physiological
• energy dependent
• controlled
• affects individual / cluster of
cells
• cell shrinkage
• pyknosis, karyorrhexis
• intact cell membrane
• cytoplasm retained in
apoptotic bodies
APOPTOSIS
NECROSIS VS APOPTOSIS

24. ASSAYS OF APOPTOSIS
1. Cytomorphological alterations
2. DNA fragmentation
3. Detection of Caspases, cleaved substrates, regulators
and inhibitors
4. Membrane alterations
5. Detection of apoptosis in whole mounts
6. Mitochondrial assays

25. • TECHNIQUES TO IDENTIFY APOPTOTIC CELLS
• 1.Staining of condensed chromatin by haematoxylin, Feulgen stain.
• 2.DNA changes detected by in situ techniques or by gel electrophoresis.
• 3.Immunohistochemical stains with antibody specific for caspase 3 antibody.
• 4.Fluorescent staining with annexin V to locate phosphatidylserine on the outer
surface of the cell membrane in apoptosis.
• 5.Fluorochrome-based TUNEL(terminal deoxynucleotidyl transferase dUTP end-
labelling) assay by flow cytometry for detecting DNA breakages.

26. VARIANTS OF PROGRAMMED CELL DEATH
• NECROPTOSIS:- This form of cell death is a hybrid that shares aspects of both
necrosis and apoptosis.
• Morphologically, and to some extent biochemically, it resemble necrosis, as both
are characterised by loss of ATP, swelling of the cell and organelles, generation of
reactive oxygen species(ROS), release of lysosomal enzymes, and ultimately rupture
of the plasma membrane.
• Mechanistically, it is triggered by signal transduction pathways that culminate in
cell death, a feature similar to apoptosis.
• It is a ‘’caspase –independent’’ programmed cell death.
• E.g.Physiological necroptosis-occurs during formation of mammalian bone growth
plate.
• Pathologically- steatohepatitis, acute pancreatitis, ischemia-reperfusion injury, viral

27. • PYROPTOSIS:- is a form of apoptosis that is accompanied by the release of the
fever-inducing cytokine IL-1
• Microbial products that enter infected cells are recognized by cytoplasmic innate
immune receptors and can activate the multiprotein complex called the
inflammasome, to activate caspase-1, which cleaves a precursor form of IL-1 and
releases its biologically active form.
• Pyroptosis differs from classic apoptosis in having
• 1. Cellular swelling
• 2. Damage to plasma membrane
• 3. By release of fever-producing cytokine IL-1

28. • FERROPTOSIS:- Ferroptosis is cell death triggered by iron-
dependent accumulation of reactive oxygen species in the
cell.
• The peroxidation of lipids disrupts many aspects of membrane
function, including fluidity, lipid-protein interactions, ion and
nutrient transport, and signaling pathways.
• The overall effect is the loss of membrane permeability, which
leads to cell death resembling necrosis but is regulated by
specific signals (unlike necrosis) and can be prevented by
reducing iron levels
• E.g. cancers
• neurodegenerative diseases
• stroke

29. CLINICAL APPLICATIONS
• 1. Conditions with defective apoptosis and increased cell survival
Mutation of TP53 is most common in various human cancers like cancers of the
lung, head and neck, colon and breast
Autoimmune diseases, immunodeficiencies, lymphoid malignancies.
2.conditions with exaggerated apoptosis and higher cell death
In Acute myocardial infarction in which there is 20% necrosis and 80% apoptosis
of infarcted myocardium
In Neurodegenerative diseases (e.g.Alzheimer’s disease, parkinson’s disease),
misfolded proteins and mutations cause increased apoptosis
Hematological diseases such as myelodysplastic syndromes, aplastic anemia,
severe beta thalassaemia

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