Apoptosis — AP 22 (15 Marks)

A. Definition of Apoptosis

• The plasma membrane remains intact throughout, but is altered to display "eat-me" signals (e.g., phosphatidylserine flip to outer leaflet)
• The cell fragments into membrane-bound apoptotic bodies, which are rapidly phagocytosed by macrophages
• No inflammatory reaction is elicited because cellular contents do not leak into the extracellular space (contrast with necrosis)
• It occurs in both physiologic (normal development, immune homeostasis) and pathologic conditions (DNA damage, viral infections)

"Apoptosis is a pathway of cell death in which cells activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins." — Robbins & Kumar Basic Pathology

Morphologic Features of Apoptosis

B. Pathways of Apoptosis (Mechanisms in Detail)

1. Mitochondrial (Intrinsic) Pathway

• Withdrawal of growth factors/survival signals
• DNA damage (radiation, toxins, free radicals)
• Accumulation of misfolded proteins (ER stress)
• Oxidative stress

• Anti-apoptotic proteins: BCL-2 and BCL-X_L are produced in response to growth factors; they maintain mitochondrial membrane integrity by holding BAX/BAK in check.
• Pro-apoptotic proteins: BAX and BAK are two effectors that, when freed from BCL-2/BCL-X_L inhibition by BH3-only proteins, dimerize and insert into the outer mitochondrial membrane, forming channels.

• Activate a DNase (CAD) that degrades nuclear DNA into oligonucleosomal fragments → "DNA ladder" on gel electrophoresis
• Proteolyze structural components of the nuclear matrix → nuclear fragmentation
• Break down cytoskeletal proteins → cell shrinkage and blebbing

2. Death Receptor (Extrinsic) Pathway

• TNFR1 (Type 1 TNF receptor)
• Fas (CD95) — the best-characterized death receptor

• FasL (Fas ligand) — expressed on cytotoxic T lymphocytes (CTLs) and self-reactive T cells
• TNF-α

3. Convergence of Both Pathways — The Execution Phase

1. Nuclear DNA fragmentation (by CAD endonuclease)
2. Nuclear membrane dissolution
3. Cytoskeletal breakdown
4. Membrane blebbing → apoptotic bodies
5. Phosphatidylserine exposure → "eat-me" signal for phagocytes

4. Role of p53 in Apoptosis

• DNA damage → stabilizes p53 (tumor suppressor protein)
• p53 upregulates BAX and BH3-only proteins (e.g., PUMA, NOXA)
• This tilts the BCL-2 balance toward pro-apoptotic proteins → intrinsic pathway activation
• Loss of p53 (e.e., mutation in cancer cells) allows cells with damaged DNA to survive → carcinogenesis

Summary Diagram of Intrinsic Pathway:

Growth factor withdrawal / DNA damage / Misfolded proteins
         ↓
  BH3-only proteins activated (PUMA, NOXA, BIM)
         ↓
  BCL-2/BCL-XL inhibited → BAX/BAK freed
         ↓
  BAX/BAK dimerize → insert into outer mitochondrial membrane
         ↓
  Cytochrome c + Smac/DIABLO released into cytosol
         ↓
  Cytochrome c + APAF-1 → APOPTOSOME
         ↓
  Caspase-9 activated (initiator)
         ↓
  Caspase-3 / Caspase-6 activated (executioners)
         ↓
  DNA fragmentation, nuclear collapse, apoptotic bodies

C. Other Methods of Cell Death

1. Necroptosis ("Programmed Necrosis")

• Definition: A form of regulated cell death that morphologically resembles necrosis but is genetically programmed (like apoptosis)
• Triggers: Ligation of TNFR1, viral RNA/DNA sensors (e.g., RIG-I, DAI), and other ligands
• Mechanism: Activated by a kinase called RIPK3 (receptor-interacting protein kinase 3), which phosphorylates MLKL (mixed lineage kinase domain-like protein) → MLKL inserts into the plasma membrane → loss of membrane integrity → cell swelling and rupture
• Key feature: Occurs when caspases are inhibited (e.g., in viral infections where the virus blocks caspase-8)
• Significance: Releases DAMPs (damage-associated molecular patterns) → triggers inflammation; may contribute to ischemia-reperfusion injury, neurodegeneration, inflammatory bowel disease

2. Pyroptosis ("Fiery Death")

• Definition: A highly inflammatory form of programmed cell death associated with activation of caspase-1
• Triggers: Intracellular danger signals (e.g., cytosolic LPS, uric acid crystals, bacterial toxins) detected by NOD-like receptors (NLRs)
• Mechanism:
  • Activated NLRs → assembly of the inflammasome (multiprotein complex)
  • Inflammasome activates caspase-1 → cleaves pro-IL-1β and pro-IL-18 into active, secreted forms
  • Caspase-1 also cleaves gasdermin D → N-terminal fragment inserts into the plasma membrane → pore formation → cell swelling and lysis
  • Also activated by caspase-4/5 (human) in response to intracellular LPS
• Significance: Important in innate immune defense; contributes to the pathogenesis of gout, atherosclerosis, and septic shock

3. Ferroptosis ("Iron-dependent Death")

• Definition: A form of cell death driven by iron-dependent accumulation of oxidized lipids (lipid peroxidation) — morphologically and mechanistically distinct from apoptosis and necrosis
• Triggers: Depletion of glutathione peroxidase 4 (GPX4), which normally neutralizes lipid peroxides; excess intracellular iron
• Mechanism:
  • Iron (via Fenton reaction) + polyunsaturated fatty acids → lipid peroxides accumulate
  • GPX4 normally detoxifies these; when GPX4 is inhibited (e.g., by erastin/RSL3), peroxides accumulate → membrane damage → cell death
• Morphology: Shrunken mitochondria with increased membrane density; no nuclear fragmentation (distinguishes it from apoptosis)
• Significance: Implicated in neurodegeneration, acute kidney injury, ischemia-reperfusion injury, and cancer cell death

4. Autophagy-Associated Cell Death

• Definition: Autophagy is primarily a survival mechanism (degradation of damaged organelles); however, excessive autophagy can lead to cell death
• Mechanism: Cellular contents are engulfed in autophagosomes → fuse with lysosomes → contents degraded; when overwhelming, leads to cell death
• Significance: Implicated in neurodegeneration, cancer, starvation-induced cell death

Summary Table — Cell Death Mechanisms

Key Points for Exam (KNRUHS)

1. Apoptosis = programmed, non-inflammatory, energy-dependent cell death; membrane remains intact
2. Two major pathways: Intrinsic (mitochondrial/BCL-2/caspase-9) and Extrinsic (death receptor/Fas-FADD/caspase-8)
3. Both converge on caspase-3/6 (executioners) → DNA ladder + apoptotic bodies
4. p53 links DNA damage to apoptosis via BH3-only proteins
5. Other cell death mechanisms: Necroptosis (RIPK3/MLKL), Pyroptosis (caspase-1/gasdermin D), Ferroptosis (GPX4/lipid peroxides)