Cell Death Notes

Cell Death

Learning Objectives

• Revise necrosis versus apoptosis.

• Understand key pathways leading to apoptosis.

  • Importance of balance between pro- and anti-apoptotic factors, especially the BCL2 family.

    • Extrinsic: receptor-mediated.

    • Intrinsic: intracellular stress, T cell, DNA damage, p53.

• Know what caspases are.

• Know why apoptotic cells are phagocytosed.

• Be aware of other variants of cell death such as anoikis, pyroptosis, ferroptosis, necroptosis.

Reversible Cell Injury

• Plasma membrane blebbing.

• Increased intracellular volume.

• Disaggregated ribosomes.

• Dilated, vesicular endoplasmic reticulum.

• Mitochondrial swelling and calcification.

• Aggregated cytoskeletal elements.

Oxygen Free Radicals & Mitochondria

• Production and detoxification of reactive oxygen species (ROS).

• Enzymes involved:

  • P450 enzymes.

  • Electron transport chain.

  • Superoxide dismutase (SOD): 2O2 + 2H^+ \rightarrow H2O2 + O2

  • Glutathione peroxidase (GPX): H2O2 + 2GSH \rightarrow 2H_2O + GSSG

  • Catalase: 2H2O2 \rightarrow O2 + 2H2O

Lethal Injury

• Ischemia: Lack of blood flow (e.g., due to thrombus in a coronary artery).

• Consequences of ischemia:

  • Decreased oxygen and glucose supply.

  • Increased anaerobic glycolysis, leading to lactate production.

  • Decreased ATP production.

  • Failure of Na+/K+ exchange, leading to intracellular K+ decrease and Na+ increase.

  • Increased Na+/H+ exchange, leading to intracellular H+ increase.

  • Increased Na+/Ca2+ exchange, leading to intracellular Ca2+ increase.

  • Activation of phospholipases, causing cell membrane damage.

  • Necrosis.

Necrosis

• Definition: Death of tissues following bioenergetic failure and loss of plasma membrane integrity.

• Induces inflammation and repair.

• Causes: Ischemia, metabolic issues, trauma.

• Types:

  • Coagulative necrosis: Firm, pale area with ghost outlines on microscopy (most tissues).

  • Colliquative necrosis: Liquefied dead area (brain).

  • Caseous necrosis: Pale yellow, semi-solid material (tuberculosis).

  • Gangrene: Necrosis with putrefaction, often black, following vascular occlusion or certain infections.

  • Fibrinoid necrosis: Microscopic feature in arterioles in malignant hypertension.

  • Fat necrosis: May follow trauma (causing a mass) or pancreatitis (visible as multiple white spots).

• Pathological process.

• Often affects a solid mass of tissue.

• Evokes an inflammatory response.

Apoptosis

• Programmed cell death involves fragmentation.

• Roles:

  • Embryology (lumen formation in tubes).

  • Response to growth signals (menstrual cycle).

  • Inflammation resolution (death of neutrophils).

  • Immune defense (T and Natural Killer cell responses).

  • Tumor prevention (preventing mutation).

  • Autoimmune disease (self-destruction).

  • HIV/AIDS (HIV and activated T cell death).

Apoptosis: DNA Fragmentation

• Apoptosis usually involves DNA fragmentation.

• Key players:

  • CAD (Caspase-Activated DNase).

  • ICAD (Inhibitor of CAD).

  • Caspases (especially Caspase 3).

  • PARP (Poly (ADP-ribose) polymerase).

  • p53.

• Process:

  • Caspases cleave ICAD, activating CAD.

  • CAD degrades DNA into fragments.

  • PARP is cleaved by caspases, preventing DNA repair.

Apoptosis: Phagocytosis

• Recognition by macrophages and non-professional phagocytes.

• Clearance requires reorganisation of phosphatidylserine (PS).

  • Healthy cells: PS in the inner leaflet of the cell membrane.

  • Apoptotic cells: PS translocates to the outer leaflet, signaling phagocytosis.

Apoptosis Pathways

• Extrinsic (Receptor-mediated):

  • TNF family (e.g., TNF, Fas/CD95).

  • T cells.

• Intrinsic (Stress-induced):

  • Metabolic stress.

  • DNA damage and p53 activation.

Extrinsic Pathway

• Receptor interaction.

• Cytoplasmic signals.

• Caspase cascade.

• Example: TNF signaling

  • TNF binds to TNFR (TNF receptor).

  • Recruits adaptor proteins with death domains.

  • Activates procaspase-8.

  • Caspase-8 activates effector caspases (Caspases-3, 6, 7).

  • Effector caspases cleave target proteins (endonucleases like PARP, nuclear proteins like lamin, cytoskeletal proteins).

T Cell-Mediated Extrinsic Pathway

• T cell releases perforin and granzymes.

• Perforin creates pores in the target cell membrane.

• Granzymes enter the target cell and activate procaspase-10, initiating apoptosis.

• Examples: Viral infection, transplantation rejection, tumor cell destruction.

Intrinsic Pathway

• Endogenous activation.

• Mitochondrial involvement.

• Process:

  • Intracellular stress triggers release of cytochrome c from mitochondria.

  • Cytochrome c binds to Apaf-1 (adaptor protein), forming an apoptosome.

  • The apoptosome recruits and activates procaspase-9.

  • Activated caspase-9 initiates the caspase cascade.

Intrinsic Pathway: Cytochrome C Release

• Release of cytochrome c from mitochondria is a key step.

• Regulated by the BCL2 family of proteins.

Apoptosome

• Formation and function:

  • Cytochrome c released from mitochondria binds to Apaf-1.

  • Apaf-1 oligomerizes to form the apoptosome.

  • Procaspase-9 is recruited to the apoptosome and activated.

  • Activated caspase-9 initiates the caspase cascade, leading to apoptosis.

DNA Damage and p53

• DNA damage activates p53.

• Activated p53:

  • Induces cell cycle arrest (G1/S stop) via p21WAF/CIP1.

  • Promotes apoptosis by increasing the production of pro-apoptotic proteins (e.g., PUMA, NOXA) and increasing Bax.

  • Bax opens the mitochondrial permeability transition pore (MPTP), leading to cytochrome c release.

Controls of Apoptosis: BCL2 Family

• Balance between pro- and anti-apoptotic factors is crucial.

• BCL2 family members:

  • Anti-apoptotic: Bcl-2, Bcl-XL.

  • Pro-apoptotic: Bax, Bak, Bim, Bad, Noxa.

• Mechanism:

  • Activated p53 increases the production of pro-apoptotic proteins like Bim and Noxa.

  • Activator pro-apoptotic proteins outcompete Bax and Bak for anti-apoptotic binding.

  • Liberated Bax and Bak open the mitochondrial transition permeability pore, releasing cytochrome c.

BCL-2 Family Proteins

• (A) Anti-apoptotic (e.g., Bcl2, Bcl-Xl)

• (B) Pro-apoptotic BH123 protein (e.g., Bax, Bak)

• (C) Pro-apoptotic BH3-only protein (e.g., Bad, Bim, Bid, Puma, Noxa)

Abnormal BCL2 Expression

• Abnormal BCL2 expression can contribute to cancer, e.g., Follicular Lymphoma.

• Translocation and rearrangement between chromosomes 14 and 18.

• t(14;18)

Multiple Layers of Control

• Inactive intrinsic pathway:

  • Cytochrome c blocked.

  • Procaspases inactive.

• Activation of Intrinsic Pathway:

  • Apoptotic stimulus.

  • Activated BH123 proteins.

  • Apoptosome formation.

  • Activated caspase-9.

Caspases: Key Effectors of Apoptosis

• Cysteine proteases that cleave after aspartate residues.

• Phenotypic changes in cells require cleavage of cellular proteins by caspases:

  • Cleavage of ICAD leads to DNA degradation.

  • Cleavage of PARP prevents DNA repair.

  • Cleavage of lamin breaks down nuclear architecture.

  • Cleavage of keratin breaks down cytoplasmic architecture.

Caspases: A Cascade

• Procaspases are activated by cleavage.

• Active caspase consists of large and small subunits.

Intrinsic and Extrinsic Pathway Links

• The two pathways are not independent; they communicate.

• Survival factors can override apoptosis.

• Importance in carcinogenesis.

Intrinsic and Extrinsic Pathways Communicate

• Initiator procaspases (e.g., caspase 8, caspase 9, caspase 10) are activated by death ligands or intracellular signals.

• Executioner procaspases (e.g., caspase 3) are activated by initiator caspases.

• Executioner caspases cleave substrates like ICAD, lamin, vimentin, actin, which lead to cell death.

Survival Factors

• Mechanisms by which survival factors block apoptosis:

  • Increased production of anti-apoptotic Bcl2 protein.

  • Inactivation of pro-apoptotic BH3-only Bcl2 protein (e.g., Bad).

  • Inactivation of anti-IAPs.

Calorie Restriction and Lifespan

• Calorie restriction lengthens lifespan through various mechanisms:

  • Reduced insulin and IGF-I signaling.

  • Activation of Sirt1.

  • Decreased mTOR activity.

  • Increased Ku70 activity.

  • Activation of FOXO transcription factors.

  • Increased DNA repair and antioxidant production.

Aberrant Apoptosis Effects -

• When apoptosis goes wrong:

  • Autoimmune disease.

  • Cancer.

  • Neurodegeneration.

• Potential drug targets: Bcl2 in lymphoma, Caspase 3 in Alzheimer Type Dementia, IAP in cancer.

Other Cell Death Types: Pyroptosis

• Microbial trigger (e.g., Salmonella).

• Pattern recognition receptors (NOD-like and Toll-like receptors).

• Features similar to both apoptosis and necrosis:

  • Caspase 1 activation (not caspase 3).

  • Nuclear fragmentation but not cytoplasmic blebbing.

  • Pro-inflammatory.

Other Cell Death Types: Anoikis

• Death after losing contact with basement membrane/extracellular matrix.

• Apoptosis morphology.

• Relevance to metastasis.

Learning Objectives Review

• Revise necrosis versus apoptosis

• Understand key pathways leading to apoptosis and the importance of pro- and anti-apoptotic factor balance:

  • Extrinsic: receptor-mediated

  • Intrinsic: intracellular stress, T cell, DNA damage, p53

• Know what caspases are

• Understand why apoptotic cells are phagocytosed

• Be aware of other variants of cell death (anoikis, pyroptosis, ferroptosis, necroptosis)