Apoptosis Detailed Notes

Cell Proliferation, Survival, and Death

• Cell proliferation, cell survival, and cell death are fundamental processes. Apoptosis is a key mechanism of cell death.

Types of Cell Death

• Three major morphologically distinct types of cell death:

  • Apoptosis (Type I cell death)

  • Autophagic cell death (Type II)

  • Necrosis (Type III)

Apoptosis

• Apoptosis, from the Greek for "falling leaves," is a natural biochemical process of cell death.

• Maintains homeostasis: Approximately 3 billion cells are born each minute, and a corresponding 3 billion undergo apoptosis to maintain balance.

• Essential for restructuring tissues and organs during embryogenesis and development.

Initiation of Apoptosis

• Apoptosis can be triggered by various signals:

  • Damaged DNA.

  • A cell entering the S phase under improper conditions.

  • Lack of proper contact with the extracellular matrix.

  • Lack of necessary growth factors.

  • Presence of death signal proteins.

Characteristics of Apoptosis

• Apoptosis is characterized by:

  1. Activation of caspase proteases.

  2. Cell shrinkage.

  3. Membrane blebbing.

  4. Condensation of chromatin (pyknosis).

  5. Activation of endonucleases that degrade chromosomal DNA into nucleosome-size fragments (approximately 170 base pairs).

  6. Engulfment and digestion of cell remnants by phagocytic cells like macrophages.

Caspases

• Caspase-generated changes affect the plasma membrane, cytoskeleton, and nuclear DNA.

• Caspases are cysteine proteases with specificity for aspartic acid residues in their substrates.

Executioner Caspases

• Executioner caspases (caspase-3, -6, and -7) are responsible for destruction and are produced as inactive dimers lacking protein-interaction domains.

Initiator Caspases

• Initiator caspases (caspase-2, -8, and -9) are activated by induced proximity or cleavage.

Threat to Surrounding Cells

• Apoptotic cells do not pose a threat to surrounding cells.

• Changes in their plasma membranes facilitate rapid phagocytosis by macrophages and neighboring cells.

• Macrophages that phagocytose apoptotic cells do not release cytokines that initiate the inflammatory response.

Major Pathways of Apoptosis

• Death receptor pathway (extrinsic pathway).

• Mitochondrial pathway (intrinsic pathway).

Death Receptor Pathway (Extrinsic)

• Apoptosis is triggered when cell-surface death receptors, like Fas, are bound by their ligands.

• Death ligand binding promotes binding of intracellular adaptor proteins to the cytoplasmic region of the receptor.

• Adaptor proteins aggregate through death domain (DD) interactions, including pro-caspase 8.

• Pro-caspase 8 acquires auto-proteolytic activity, cleaving peptide bonds to generate active caspase 8.

• Caspase 8 initiates the protease caspase cascade.

Extrinsic Pathway Details

• Two pathways:

  • Ligand-receptor interactions (FasL binding to Fas [CD95] or TNF-$\alpha$ binding to its receptor).

  • Immune cell (cytotoxic T-cell release of perforin and granzyme B).

• Fas-FasL interaction is necessary for thymic medullary negative selection.

• Mutations in Fas lead to increased numbers of circulating self-reacting lymphocytes due to failure of clonal deletion.

• Defective Fas-FasL interactions cause autoimmune lymphoproliferative syndrome.

Intrinsic Pathway (Mitochondrial)

• Involved in tissue remodeling during embryogenesis.

• Occurs when a regulating factor is withdrawn from a proliferating cell population (e.g., IL-2 after a completed immunologic reaction leading to apoptosis of proliferating effector cells).

• Also occurs after exposure to injurious stimuli (e.g., radiation, toxins, hypoxia).

• Regulated by the Bcl-2 family of proteins.

• BAX and BAK are proapoptotic, while Bcl-2 and Bcl-x are antiapoptotic.

• Bcl-2 keeps the mitochondrial outer membrane impermeable, preventing cytochrome c release.

• Bcl-2 overexpression (e.g., follicular lymphoma t[14;18]) inhibits caspase activation, promoting tumorigenesis.

Apoptosome Formation

• In the cytoplasm, cytochrome c binds to a protein aggregate containing multiple molecules of the adaptor protein Apaf1 (apoptotic protease-activating factor 1) and procaspase 9.

• This complex is called the apoptosome.

• Cytochrome c addition causes autocatalytic hydrolysis of peptide bonds in pro-caspase 9, resulting in active caspase 9.

• Caspase 9 activates the caspase cascade to execute apoptotic death.

Cellular Events - DNA Fragmentation

• DNA fragmentation occurs in the nucleus, irreversibly committing the cell to die.

• Result of $Ca^{2+}$-dependent and $Mg^{2+}$-dependent activation of nuclear endonucleases.

• These enzymes selectively cleave DNA, generating small oligonucleosomal fragments.

• Nuclear chromatin aggregates, and the nucleus may divide into discrete fragments.

Cellular Events - Decrease in Cell Volume

• Achieved by shrinking of the cytoplasm.

• Cytoskeletal elements reorganize into bundles parallel to the cell surface.

• Ribosomes clump, the rER forms concentric whorls, and endocytotic vesicles fuse with the plasma membrane.

Cellular Events - Loss of Mitochondrial Function

• Caused by changes in the permeability of mitochondrial membrane channels.

• Mitochondrial integrity is breached, the transmembrane potential drops, and the electron-transport chain is disrupted.

• Proteins from the mitochondrial intermembrane space, like cytochrome C, are released into the cytoplasm.

• Cytochrome C activates caspases, dismantling the cell.

• Mitochondria, influenced by Bcl-2 proteins, are decision-makers for initiating apoptosis.

Cellular Events - Membrane Blebbing

• Results from cell membrane alterations.

• Translocation of molecules (e.g., phosphatidylserine) from the cytoplasmic surface to the outer surface of the plasma membrane.

• Causes changes in physical and chemical properties, leading to blebbing without loss of membrane integrity.

Cellular Events - Formation of Apoptotic Bodies

• The final step of apoptosis, resulting in cell breakage.

• Membrane-bounded vesicles originate from cytoplasmic blebs containing organelles and nuclear material.

• Rapidly removed by phagocytotic cells without an inflammatory response.

• Occurs more than 20 times faster than mitosis, making apoptotic cells challenging to find in routine H&E preparations.

p53 Regulated Cell Cycle

• Negative regulation of the cell cycle by intracellular signals.

• Checkpoints block cells from proceeding through the cell cycle if damaged.

• p53 detects DNA damage and activates p21.

• p21 inhibits Cdk2-cyclinA, arresting the cell cycle at G1 or G2 phases to allow DNA repair.

p53 Induction of Apoptosis

• Cells with damaged DNA are stopped from completing cell division by p53-dependent mechanisms.

• p53 increases levels of CKI p21 to inhibit Cdk-promoted progression through the cell cycle at the G1-S or G2-M phase transitions.

• If DNA damage is irreparable, p53 initiates cellular apoptosis.

• Elimination of cells with DNA damage prevents tumor development.

• Loss of p53 regulation of cellular apoptosis is common in cancers.

p53-Induced Apoptosis

• If DNA damage is too great to repair, higher concentrations of p53 result in p53-induced apoptosis by:

  • Increasing the transcription rates of Bax and pro-apoptotic proteins like PUMA.

  • Activating cytoplasmic Bax, causing it to associate in the mitochondrial membrane.

Examples of Apoptosis

• Menstrual shedding of endometrium (apoptosis induced when estrogen and progesterone levels decrease).

• Destruction of specific cells during embryogenesis (loss of Mullerian structures in males due to Mullerian inhibitory factor).

• Virally infected cells (apoptosis induced by cytotoxic T-cells).

• Embryogenesis (apoptosis induced in the skin between fingers).

Anoikis

• A form of apoptosis induced by a lack of cell-to-extracellular matrix interactions.

• Cell-detachment-induced apoptosis prevents detached cells from further growth and reattachment to an inappropriate extracellular matrix.

• Under these conditions, the cell cycle is arrested, and apoptosis is initiated.

• Signals from an intercellular matrix are sensed by integrins, which are involved in signaling mechanisms that control apoptosis, DNA damage responses, and the function of death receptors.

• Defects in these signaling pathways lead to anoikis, which is triggered by the activation of the proapoptotic Bcl-2 family of proteins.

• Anoikis leads to cytochrome C release, activating caspase enzymes and initiating apoptosis.

Metastatic Cancer and Anoikis

• In metastatic cancer, cells develop mechanisms to survive the anoikis process.

• This resistance is due to various mechanisms, including:

  • Changes in integrin receptor types.

  • Activation of antiapoptotic factors.

  • Oncogene activation.

  • Growth factor receptor signaling.

Diseases Related to Apoptosis

• Defects in programmed cell death contribute to major diseases.

  • Excessive apoptosis causes extensive nerve cell loss in Alzheimer's disease and stroke.

  • Insufficient apoptosis has been linked to cancer and autoimmune diseases.

Apoptosis vs. Necrosis

• Necrosis begins with impairment of the cell’s ability to maintain homeostasis.

• Cell injury leads to damage to the cell membrane and influx of water and extracellular ions.

• Intracellular organelles undergo irreversible changes caused by cell swelling and membrane rupture (cell lysis).

• Cytoplasmic contents, including lysosomal enzymes, are released into the extracellular space.

• Necrotic cell death is often associated with extensive surrounding tissue damage and an intense inflammatory response.

Features Distinguishing Necrosis from Apoptosis

• Necrosis involves cell swelling and damage to the plasma membrane; apoptosis involves cell shrinkage and plasma membrane blebbing.

• Necrosis leads to random DNA degradation, while apoptosis leads to oligonucleosomal DNA fragmentation.

• Apoptosis involves caspase cascade activation, which is absent in necrosis.