Molecular basis of apoptosis mechanism and morphological changes md pathology exam question Robbins based answer
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Robbins Basic Pathology apoptosis intrinsic extrinsic pathway morphology caspases
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Apoptosis: molecular basis, mechanism and morphological changes
Definition:
Apoptosis is a regulated, energy-dependent form of cell death in which a cell activates enzymes that degrade its own nuclear DNA and cytoplasmic proteins. The cell fragments into membrane-bound apoptotic bodies, which are rapidly phagocytosed without leakage of cellular contents or inflammation.
Robbins & Kumar Basic Pathology, p. 24.
Apoptosis is a regulated, energy-dependent form of cell death in which a cell activates enzymes that degrade its own nuclear DNA and cytoplasmic proteins. The cell fragments into membrane-bound apoptotic bodies, which are rapidly phagocytosed without leakage of cellular contents or inflammation.
Robbins & Kumar Basic Pathology, p. 24.
Molecular basis
The central mediators are caspases: cysteine proteases that cleave proteins after aspartate residues. They are synthesized as inactive procaspases and become activated in a cascade.
- Initiator caspases: caspase-8, caspase-9 (and caspase-10)
- Executioner caspases: caspase-3, caspase-6, caspase-7
Apoptosis proceeds through two main pathways that converge on executioner caspase activation.
1. Intrinsic or mitochondrial pathway
This is the major pathway in most physiological and pathological apoptosis.
Stimuli
- Withdrawal of growth factors or hormones
- DNA damage by radiation, cytotoxic drugs, hypoxia, or oxidative stress
- Accumulation of misfolded proteins causing ER stress
- Severe cellular injury
Mechanism
- Cellular stress activates BH3-only proteins such as Bim, Bid, Bad, Puma, and Noxa.
- BH3-only proteins inhibit anti-apoptotic BCL-2 family members:
- BCL-2
- BCL-XL
- MCL-1
- This permits activation and oligomerization of pro-apoptotic proteins BAX and BAK in the outer mitochondrial membrane.
- BAX and BAK cause mitochondrial outer membrane permeabilization.
- Cytochrome c is released into cytosol.
- Cytochrome c combines with Apaf-1 and procaspase-9 to form the apoptosome.
- Apoptosome activates caspase-9, which activates executioner caspases, especially caspase-3 and caspase-7.
- Executioner caspases produce the structural and nuclear changes of apoptosis.
Flow chart:
DNA damage / growth factor withdrawal / ER stress→
BH3-only proteins→ inhibition of
BCL-2, BCL-XL→ activation of
BAX and BAK→ mitochondrial permeability
→
cytochrome c release→
Apaf-1 apoptosome→
caspase-9→
caspase-3, -6, -7→ apoptosis
Robbins & Kumar Basic Pathology, p. 24.
2. Extrinsic or death receptor pathway
This pathway is initiated by extracellular death signals acting through cell-surface death receptors of the TNF receptor family.
Important receptors and ligands
- Fas (CD95) receptor and Fas ligand (FasL)
- TNF receptor 1 and TNF
- TRAIL receptors
Mechanism
- FasL on activated cytotoxic T cells binds Fas/CD95 on the target cell.
- Fas receptors aggregate and recruit adaptor proteins, especially FADD (Fas-associated death domain).
- This produces the death-inducing signaling complex (DISC).
- DISC activates initiator caspase-8.
- Caspase-8 activates executioner caspases, mainly caspase-3 and caspase-6.
- The target cell undergoes apoptosis.
Flow chart:
FasL or TNF→
Fas/CD95 or TNF receptor→
FADD + DISC formation→
caspase-8→
executioner caspases→ apoptosis
Caspase-8 can also cleave Bid, a BH3-only protein, thereby amplifying apoptosis through the mitochondrial pathway.
Robbins & Kumar Basic Pathology, p. 24.
3. Cytotoxic T-cell-mediated apoptosis
Cytotoxic T lymphocytes and NK cells kill virus-infected, tumor, and graft cells by:
- FasL-Fas interaction, and
- Release of perforin and granzyme B.
Perforin forms pores in the target-cell membrane, allowing granzyme B to enter and activate caspases directly. This produces apoptosis.
Execution phase: how caspases cause cell death
Activated executioner caspases cause:
-
Cleavage of cytoskeletal and nuclear proteins
Causes cell shrinkage, membrane blebbing, and fragmentation. -
Activation of endonucleases
Caspase-3 cleaves ICAD, the inhibitor of caspase-activated DNase (CAD).
Free CAD fragments DNA into nucleosomal units, producing the characteristic DNA ladder pattern. -
Inactivation of DNA repair proteins
For example, cleavage of PARP prevents DNA repair. -
Alteration of plasma membrane
Phosphatidylserine becomes exposed on the outer membrane surface, acting as an “eat-me” signal for macrophages and adjacent cells.
Morphological changes in apoptosis
1. Cell shrinkage
- The cell becomes smaller.
- Cytoplasm becomes dense and intensely eosinophilic.
- Organelles remain relatively intact but are closely packed.
2. Chromatin condensation
- This is the most characteristic morphologic feature.
- Chromatin aggregates beneath the nuclear membrane as dense peripheral crescents.
- The nucleus may later fragment into multiple pieces, called karyorrhexis.
3. Membrane blebbing
- The plasma membrane remains intact but forms protrusions or blebs.
- There is no early loss of membrane integrity.
4. Formation of apoptotic bodies
- The cell breaks into small, membrane-bound fragments.
- These contain cytoplasm, organelles, and sometimes nuclear fragments.
5. Rapid phagocytosis
- Macrophages or neighboring parenchymal cells engulf apoptotic bodies.
- Lysosomal enzymes degrade them.
6. Absence of inflammation
- Cellular constituents do not leak into extracellular tissue.
- Therefore, apoptosis typically does not cause an inflammatory reaction.
Appearance on H&E stain
An apoptotic cell appears as a round or oval shrunken cell with:
- Intensely eosinophilic cytoplasm
- Condensed, dark basophilic nuclear chromatin
- Nuclear fragments or apoptotic bodies
Robbins, Cotran & Kumar Pathologic Basis of Disease, Fig. 2.15.
Apoptosis versus necrosis
| Feature | Apoptosis | Necrosis |
|---|---|---|
| Cell size | Shrinkage | Swelling |
| Cell membrane | Intact, with blebs | Disrupted and ruptured |
| Nuclear change | Condensation and fragmentation | Pyknosis, karyorrhexis, then karyolysis |
| Cell contents | Retained within apoptotic bodies | Leak into extracellular space |
| Inflammation | Absent or minimal | Marked inflammation |
| Mechanism | Caspase-mediated programmed death | Severe injury with membrane damage and ATP depletion |
One-line conclusion
Apoptosis is caspase-mediated programmed cell death occurring through intrinsic mitochondrial or extrinsic death-receptor pathways, morphologically characterized by cell shrinkage, chromatin condensation, membrane blebbing, apoptotic-body formation, rapid phagocytosis, and no inflammation.
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