Programmed Cell Death & Apoptosis

Introduction to Programmed Cell Death

  • Lecture given by one instructor, with Catherine Skelding taking over for several weeks, before the mid-semester break.
  • A reminder to email with any specific revision requests before the mid-semester exam.
  • The instructor will return for the last four weeks of the course to discuss cell signaling.
  • The lecture will focus on Programmed Cell Death.

The Significance of Cell Death

  • Cell death is the opposite of cell proliferation.
  • It is a protective mechanism against older, damaged cells that could become cancerous.
  • Cell death is essential throughout development, eliminating cells no longer needed after fulfilling their function in forming organs.
  • Apoptosis is a programmed, controlled, and regulated process, not a spontaneous event.

Learning Objectives

  • Define apoptosis as a key programmed cell death pathway, researched extensively in Australia.
  • Describe the biochemical and morphological changes in apoptotic cells (hallmark features).
  • Compare apoptosis with necrosis and autophagy (a cell-protective mechanism).
  • Explain the mechanisms that induce apoptosis:
    • Intrinsic Pathway: Initiated within the cell, often called the mitochondrial pathway.
    • Extrinsic Pathway: Triggered by loss of signals (growth factors) or binding of ligands to death receptors on the cell surface (important in the immune system).
  • Discuss the essential role of mitochondria in apoptosis.
  • Explain the functional significance of apoptosis and consequences of its dysregulation.
  • The key concept: apoptosis is a programmed and tightly regulated form of cell death, controlling when and where it occurs.

Apoptosis: Targeted Cell Death

  • Apoptosis is a targeted cell death that occurs constantly to remove old or unnecessary cells.
  • Maintains tissue size by balancing cell regeneration.
  • Removes damaged cells, particularly those with irreparable DNA damage.
  • Drugs can induce apoptosis, which is leveraged in anticancer therapy by inducing DNA damage or affecting cellular proteins.
  • Growth factor removal can induce programmed cell death, as can loss of cell contact (anoikis).
  • Apoptosis is crucial in the immune system to clear excess lymphocytes after an infection to prevent autoimmune responses.
  • Programmed cell death is conserved across various organisms, including worms, flies, mice, and humans, and also occurs in plant cells.

Apoptosis in Development

  • Apoptosis is vital during development alongside cell proliferation and differentiation.
  • Developing Limb Bud Example:
    • Early development: cells signal bone cells to develop digits.
    • Once digits are formed, the signaling cells undergo apoptosis.
    • This leads to digit separation (digitization).
    • Apoptotic cells can be stained to visualize the process.
  • Metamorphosis Example:
    • Tadpole to frog: tail cells undergo apoptosis as they are no longer needed.

Apoptosis in Adult Tissues

  • Maintains constant cell number and controls tissue size.
  • Eliminates excess cells, such as activated lymphocytes after infection.
  • Facilitates tissue remodeling after damage (e.g., stroke, heart attack), where necrosis induces apoptosis in surrounding cells to allow repair.
  • Destroys old and damaged cells, including those with DNA damage.
  • Cells have a finite lifespan (Hayflick limit), after which apoptosis is triggered.

Characteristics of Apoptosis

  • Controlled by caspases, enzymes that cleave intracellular proteins.
  • Caspases are activated by cysteine and cleave proteins at aspartic acids.

Caspase Cascade:

  • Initiator caspases activate executioner caspases.
  • Once activated, the process is irreversible, leading to cell death.
  • Caspases degrade cellular proteins to kill the cell.
  • The regulated process ensures cells are recognized by the immune system (macrophages).

Morphological Changes:

  • Cells round up and lose adhesion.
  • Membrane blebbing occurs.
  • Cells shrink, signaling macrophages to engulf them without releasing contents.

Specific Features:

  • Cell shrinkage (condensation, retraction, rounding) driven by cytoskeleton changes.
  • Membrane blebbing while maintaining membrane integrity.
  • Release of apoptotic bodies (organelle fragments).
  • DNA fragmentation, a key marker of apoptosis.

DNA Fragmentation in Detail

  • Nuclear condensation and DNA fragmentation are hallmarks of apoptosis.
  • Endonucleases (CADs - caspase-activated DNAs) cleave DNA between histones.
  • CADs are normally inactive, bound to iCAD (inhibitor of CAD).
  • During apoptosis, caspases cleave iCAD, releasing and activating CADs to degrade DNA.

DNA Laddering:

  • Running DNA on a gel shows a laddering effect, with smaller DNA fragments migrating further.
  • This DNA ladder is a hallmark of apoptosis.

TUNEL Assay:

  • Uses antibodies to detect nicks in the DNA, marking apoptotic cells.
  • This allows for visualization and quantification of apoptosis.

Membrane Blebbing Explained

  • Plasma membrane is composed of a phospholipid bilayer with different side chains on each side.
  • Phospholipid movement is controlled by enzymes (flippases, flopases).
  • During apoptosis, scramblase is activated, scrambling phospholipids and causing membrane blebbing.
  • Caspases cleave cytoskeletal components, contributing to blebbing.
  • These changes signal macrophages to engulf the cell.

Apoptosis Pathways: Intrinsic vs. Extrinsic

  • Extrinsic Pathway: External signal (death ligand) binds to a death receptor on the cell surface, activating caspases.
  • Intrinsic Pathway: Internal signal driven by changes in the mitochondria.

Intrinsic Pathway Detail:

  • Damage to the mitochondrial membrane releases cytochrome c, which activates caspases.
  • The intrinsic pathway is also called the mitochondrial pathway.
  • Crosstalk exists between the pathways, with the extrinsic pathway amplifying the intrinsic pathway.

Caspase Activation: Initiation and Execution

  • Activation of caspases is essential for apoptosis.
  • Initiator Caspases: Caspase-8 (extrinsic) and Caspase-9 (intrinsic).
  • Executioner Caspases: Caspase-3, -6, -7.
  • One initiator caspase can activate multiple executioner caspases, amplifying the signal.

Caspase Activation Mechanism:

  • Initiator caspases are inactive monomers that require adaptor proteins to dimerize and activate.
  • The protease domain is cleaved, activating the caspase to cleave downstream executioner caspases.
  • Caspase-3 is a marker of late-stage apoptosis.
  • Executioner caspases cleave proteins needed for morphological features of apoptosis.

Mitochondrial Role in Apoptosis

  • Mitochondria are essential in the intrinsic pathway and activated downstream in the extrinsic pathway.
  • Cytochrome c release:
    • Apoptotic stimuli (DNA damage) cause the mitochondria to release cytochrome c.
    • Cytochrome c binds to apoptotic protease activating factors (APAF1), activating initiator caspases.
  • BCL2 and BACs/BAX proteins regulate cytochrome c release.
    • BCL2 is anti-apoptotic, blocking apoptosis.
    • BACs/BAX are pro-apoptotic, binding to BCL2 and regulating each other.
  • The balance of these proteins determines the cell's readiness for apoptosis.

Functions Carried out by Executioner Caspases

  • Executioner caspases coordinate the demolition of cellular structures.
  • Specific functions include:
    • Changes to the actin cytoskeleton for membrane blebbing.
    • Cleavage of focal adhesion sites.
    • Golgi fragmentation.
    • Inhibition of translation by cleaving proteins involved in translation.
    • Activation of CADs (DNA fragmentation).
    • Fragmentation of the nuclear membrane by cleaving lamin proteins.

Regulation by Pro- and Anti-Apoptotic Proteins

  • The balance between pro-apoptotic (BACs, BAX) and anti-apoptotic (BCL2) proteins regulates cell fate.
  • More pro-apoptotic proteins prime the cell for apoptosis.
  • Anastasis: rare recovery from apoptosis after caspase activation, requiring strong survival signals.

Consequences of Dysregulated Apoptosis

  • Excessive Apoptosis: Tissue death (e.g., heart attack, stroke), where inhibiting apoptosis can mitigate damage.
  • Insufficient Apoptosis: Autoimmune diseases (caused by too many activated lymphocytes) and cancer (where damaged cells fail to undergo cell death).

Apoptosis and Cancer:

  • In healthy cells, DNA damage is repaired, or apoptosis is triggered.
  • If repair fails and apoptosis also fails, cancer can occur.
  • Cancer cells resist apoptosis due to an imbalance in pro- and anti-apoptotic proteins.
  • Cancer cells overcome anoikis, further promoting survival.

Necrosis: An Unregulated Cell Death

  • Necrosis is triggered by acute stress (lack of oxygen/nutrients).
  • It is a passive process; cells swell due to osmotic pressure changes, causing the membrane to burst.
  • Cellular contents are released, triggering an inflammatory response.

Key Differences Between Necrosis and Apoptosis:

  • Necrosis: Cell membrane breaks, organelles swell, ATP is lost, no apoptotic bodies, and inflammation occurs.
  • Apoptosis: Cell shrinks, plasma membrane blebbing, apoptotic bodies form, and no inflammation occurs.

Autophagy: A Cell-Protective Mechanism

  • Autophagy involves the cell breaking down parts of itself, mediated by lysosomes.
  • It is an energy-dependent process regulated under nutrient stress, aiming to protect the cell.
  • Damaged organelles, misfolded proteins, and bacteria are engulfed by autophagosomes, which fuse with lysosomes.
  • The lysosomal enzymes cleave the proteins for recycling.
  • If stress persists, autophagy signals the mitochondria to undergo intrinsic apoptosis.

Role in Disease:

  • Decreased autophagy leads to the accumulation of misfolded proteins (tau, amyloids).
  • Increased autophagy can protect cancer cells in hypoxic regions, allowing them to recycle components and avoid apoptosis.
  • The integrated stress response links autophagy and apoptosis together.