Apoptosis Notes

Apoptosis

Overview

  • Apoptosis is a type of cell death. The word comes from Greek, meaning "falling off".
  • It is characterized by specific morphological changes and is regulated by endogenously driven mechanisms.

Distinct Forms of Cell Death

  • Apoptosis (Organized Process):
    • Occurs in response to a signal.
    • The dead cell is engulfed by a phagocytic cell.
    • Neighboring cells are not damaged.
  • Non-Specific Cell Death (Due to injury):
    • The cell swells and bursts, releasing its contents.
    • This can be damaging to neighboring cells.

Cellular Features of Apoptosis

  • The dying cell shrinks, condenses, and fragments but does not burst.
  • DNA is fragmented due to cleavage by an endonuclease.
  • Changes occur in the plasma membrane lipid composition.
  • There is a loss of electrical potential in the mitochondrial membrane.
  • The apoptotic cell is usually engulfed by surrounding cells.
  • The result is a neat cell death, rapidly cleared without causing an inflammatory response.

Biochemical Hallmarks of Apoptosis

  • DNA Fragmentation:
    • Endonuclease cleaves DNA into nucleosomal units.
    • A nucleosome is approximately 150 base pairs (bp) of DNA wrapped around a core of 8 histone proteins.
  • Extracellular Phosphatidylserine:
    • Phosphatidylserine is exposed on the outer leaflet of the plasma membrane.

Apoptosis as a Genetically Programmed Process

  • Apoptosis occurs extensively in developing and adult animals.
  • In developing vertebrates, a large proportion of neurons die shortly after they are born, even if they are healthy.

Functions of Apoptosis

  • Tissue sculpting during development.
  • Deleting structures that are no longer needed.
  • Adjusting cell numbers during development.
  • Eliminating dangerous or damaged cells.
    • Examples include eliminating immune cells that recognize self-antigens or immune cells activated by infection after they have destroyed the responsible microbes.
  • Digit sculpting in the mouse paw and tube formation are examples of apoptosis.

Evidence for Factors that Inhibit Cell Death

  • Hamburger and Levi-Montalcini's work in the 1940s showed evidence of factors that inhibit cell death.

Neurotrophic Factor Hypothesis

  • Death, not life, is the default state in multicellular organisms.
  • Nerve Growth Factor (NGF) belongs to the neurotrophin family of proteins.
  • Neurotrophins bind to Trk Receptor Tyrosine Kinases (RTKs).

Genes Regulating Cell Death Discovered in Nematodes

  • Caenorhabditis elegans (C. elegans) is a nematode used in genetic studies.
    • The adult has 959 cells and is approximately 1 mm in size.
    • The complete cell lineage of C. elegans hermaphrodite has been mapped.
    • Sydney Brenner, John Sulston, and Robert Horvitz were awarded the Nobel Prize in 2002 for their work on C. elegans.
  • 131 cells undergo programmed cell death in C. elegans.
  • Apoptotic cells in C. elegans have a refractile, raised-button-like appearance.

Genetic Screens in C. elegans Identified Genes Responsible for Apoptosis

  • Cell death abnormal (ced) mutants were identified.
  • The accumulation of dead cells in ced-1 mutants occurs due to a failure to engulf dead cells.
  • Mutate and screen for phenotypes.

Phagocytosis by Mammalian Cells

  • Neutrophils are part of the innate immune system.
  • They are the most abundant type of white blood cell.
  • Neutrophils migrate to sites of inflammation and act as phagocytes to ingest microorganisms or other cells.

Genetic Screens in C. elegans Continued

  • ced-1 mutant exhibits a failure to engulf dead cells, which is used to identify animals with more or less dead cells.
  • ced-1; ced-3 double mutant shows no dead cells, indicating that the ced-3 gene is required for cell death.
  • Programmed cell death is an active biological process.
  • ced-3 and ced-4 mutants result in the survival of the 131 doomed cells.
  • ced-9 mutant causes all cells to die.
  • ced-9/ced-3 double mutants result in the survival of doomed cells.

Apoptosis Depends on a Proteolytic Cascade Mediated by Caspases

  • Caspases are synthesized as inactive dimers.
  • Amplification occurs as one initiator caspase activates many executioner caspases, and one executioner caspase acts on many substrate proteins.

Caspases Cleave Many Different Target Proteins

  • Gel electrophoresis of cells undergoing apoptosis shows a ladder of DNA fragments.
  • Endonuclease cleaves DNA into nucleosomal units.

Detection of Apoptosis

  1. Visualization of “pyknotic” nuclei (fixed tissue):

    • Example: Overexpression of dominant negative Ras in Drosophila imaginal wing disc.
    • Directly visualize fragmented DNA using GFP.

  2. TUNEL Labeling (fixed tissue):

    • TdT-mediated dUTP nick end labeling specifically labels cells with fragmented DNA.
    • Terminal deoxynucleotidyl transferase (TdT) adds labeled dUTP to the 3’ end of DNA fragments.
    • Apoptotic cells have many DNA fragments, resulting in bright fluorescence.

  3. Active Caspase-Specific Antibody (fixed tissue):

    • Cleaved caspase-3 (CC3) antibody labels apoptotic cells.

  4. Fluorescent Reporter of Apoptosis (live or fixed tissue):

    • GC3Ai: GFP sensor caspase-3-like protease activity indicator.

  5. Annexin V:

    • Binds to phosphatidylserine exposed on the outer leaflet of the plasma membrane early in apoptosis.

How is Initiator Caspase Activated?

  • Extrinsic Pathway: Activated by cell surface death receptors.
  • Intrinsic Pathway: Involves mitochondria.

Extrinsic Pathway: Activated by Cell Surface Death Receptors

  • Initiator caspase: Caspase-8.
  • Death-inducing signaling complex (DISC) is formed.
  • Many cells produce proteins that restrain the extrinsic pathway.
  • FLIP: An initiator caspase without protease activity that dimerizes with caspase-8 in DISC, preventing caspase-8 activity.
  • Killer lymphocytes (cytotoxic T cells) kill cancer cells, cells infected with a virus, or damaged cells through TNF receptors.

Intrinsic Pathway: Depends on Mitochondria

  • Depends on the release of cytochrome c from mitochondria.
  • Cytochrome c is localized between the inner and outer mitochondrial membranes and is part of the electron transport chain.
  • Cytochrome c is released from mitochondria into the cytoplasm in response to stress, DNA damage, or developmental signals.
  • Assembly of the “wheel of death”: apoptosome (caspase recruitment domain).

Bcl2 Proteins Regulate the Intrinsic Pathway of Apoptosis

  • Bcl2 (B-cell lymphoma 2) is an anti-apoptotic protein.
  • Bcl2 Homology (BH) Domains: BH4, BH3, BH1, BH2.
  • Types of Bcl2 Family Proteins:
    • Anti-apoptotic (e.g., Bcl2, BclX₁).
    • Pro-apoptotic effector (e.g., Bax, Bak).
    • Pro-apoptotic BH3-only (e.g., Bad, Bim, Bid, Puma, Noxa).

Pro-Apoptotic Effector Bcl2 Family Proteins (Bax, Bak)

  • Facilitate the release of cytochrome c from mitochondria.
  • Apoptotic stimulus causes aggregation of active effector Bcl2 family proteins, leading to cytochrome c release.

Anti-Apoptotic Bcl2 Family Proteins (Bcl2, BclX₁)

  • Prevent clustering of Bax/Bak in the mitochondrial membrane.
  • In the absence of an apoptotic signal, cytochrome c remains in the intermembrane space.

Pro-Apoptotic BH3-Only Family Proteins

  • Inhibit anti-apoptotic Bcl2 proteins and activate pro-apoptotic effector proteins.
  • An apoptotic stimulus activates BH3-only proteins, which inactivate anti-apoptotic Bcl2 family proteins.
  • This leads to the aggregation of active effector Bcl2 family proteins and the release of cytochrome c.

Different Stimuli Promote Apoptosis

  • Via Activation of Different BH3-Only Family Proteins.
  • Extrinsic apoptosis pathway activates the intrinsic pathway via the BH3-only protein BID, which amplifies the death signal.
  • p53 is activated in response to DNA damage that cannot be repaired, inducing the expression of BH3-only proteins PUMA and NOXA.
  • Cytokine signaling is required for the survival of some cell types; BH3-only proteins are activated in the absence of cytokines.

Loss of Different BH3-Only Family Proteins is Associated with Different Cancers

  • Examples:
    • BIM: Lymphoid and myeloid hyperplasia, SLE-like autoimmune disease.
    • PUMA: Resistance to γ-radiation.
    • NOXA: No major abnormalities.
    • BMF: Progressive B-lymphoid hyperplasia.
    • BAD: No major abnormalities.
    • Alterations in these genes can result in various human cancers.

Identification of Genes Required for Programmed Cell Death in Drosophila

  • Screen for genes required for normal programmed cell death during Drosophila embryogenesis.
  • Stain embryos with acridine orange, which labels apoptotic cells.
  • Mutations in genes like reaper result in no cell death.
  • reaper expression corresponds to the pattern of programmed cell death.
  • Overexpression of reaper is sufficient to induce apoptosis.
  • Additional screens identified hid and grim, which have phenotypes similar to reaper.

Inhibitor of Apoptosis Proteins (IAPs)

  • Block apoptosis induced by hid/reaper/grim (IAP antagonists).
  • Screen for genes that enhance or suppress reaper overexpression-induced apoptosis.

IAPs Block Apoptosis

  • By Binding to Activated Caspases.
  • The mammalian X-chromosome-linked IAP (XIAP) is an example.
  • IAPs bind to activated caspases via baculoviral IAP repeats (BIRs) and inhibit caspases.
  • IAP BIRs also interact with the IAP-binding motif (IBM) of IAP antagonists (e.g., reaper).
  • IAP antagonists promote apoptosis by competing with caspases for binding to IAPs.
  • The RING (really interesting new gene) domain targets caspases for ubiquitylation and degradation.

Extracellular Survival Factors Inhibit Apoptosis

  • Through Different Signaling Pathways.

Conservation of the Apoptosis Pathway

  • In C. elegans and Mammals.
  • Upstream signals lead to the activation of BH3-only proteins.
  • CED-9 in C. elegans is analogous to BCL2 in mammals.
  • CED-4 is analogous to APAF1 in mammals.
  • CED-3 is analogous to Caspase-9/Caspase-8 in mammals.

Conservation of Apoptosis Pathways

  • In Nematodes, Flies, and Mammals

Apoptosis-Induced Apoptosis

  • “Communal suicide”: Removal of large groups of cells during development.
  • Examples: Elimination of tadpole tail, removal of interdigital webbing in vertebrate hands/feet.
  • Mechanism: Studied in Drosophila wing imaginal disc.
  • Initial apoptosis induces secondary apoptosis via signaling pathways.

Apoptosis-Induced Apoptosis in Hair Follicle Cells

  • Coordinated elimination of hair follicle cells in mice.
  • Anagen (growth), Catagen (destruction), Telogen (rest).
  • In catagen, a primary apoptotic event induces a wave of secondary apoptosis that kills more hair follicle cells.
  • Apoptotic cells produce TNF, which diffuses from source cells to induce apoptosis in nearby cells.

Parallels in Cell-Death Mechanisms of Bacteria and Animals

  • Homicide Rather than Suicide.
  • Colicins: Bacterial ion-channels with structural similarity to Bcl2 family proteins.
  • Secreted by some bacterial strains, bind to receptors in the outer membrane of competing bacteria, form pores in the inner membrane, depolarize the cell membrane, and result in cell death.