Cell Biology Unit 19 ReviewApoptosis and Cell Death

What's the Big Deal?

• Apoptosis is a highly regulated and controlled process of programmed cell death that occurs in multicellular organisms
• Plays a crucial role in maintaining tissue homeostasis by eliminating old, damaged, or infected cells
• Essential for proper development and functioning of the immune system
  • Helps to remove self-reactive lymphocytes during development
  • Eliminates infected or cancerous cells during an immune response
• Involved in the prevention of cancer by removing cells with DNA damage or aberrant cell cycle regulation
• Dysregulation of apoptosis can lead to various diseases, including cancer, autoimmune disorders, and neurodegenerative diseases
• Apoptosis differs from necrosis, which is an uncontrolled and passive process of cell death caused by external factors (toxins or physical damage)
• Understanding the mechanisms of apoptosis is crucial for developing targeted therapies for diseases associated with abnormal cell death

Key Players and Pathways

• Caspases are a family of cysteine-aspartic proteases that play a central role in the execution of apoptosis
  • Initiator caspases (caspase-8, -9, and -10) are activated first and cleave and activate downstream effector caspases
  • Effector caspases (caspase-3, -6, and -7) cleave various cellular substrates, leading to the morphological and biochemical changes associated with apoptosis
• The intrinsic (mitochondrial) pathway is triggered by internal stimuli, such as DNA damage or oxidative stress
  • Regulated by the Bcl-2 family of proteins, which includes pro-apoptotic (Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) members
  • Activation leads to mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome c
  • Cytochrome c binds to Apaf-1 and procaspase-9 to form the apoptosome, which activates caspase-9 and subsequently effector caspases
• The extrinsic (death receptor) pathway is initiated by the binding of death ligands (FasL, TNF-α) to their respective death receptors (Fas, TNFR1)
  • Leads to the formation of the death-inducing signaling complex (DISC) and activation of caspase-8
  • Caspase-8 directly activates effector caspases or cleaves Bid, linking the extrinsic and intrinsic pathways
• The perforin/granzyme pathway is used by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to induce apoptosis in target cells
  • Perforin forms pores in the target cell membrane, allowing granzymes to enter the cell
  • Granzymes, particularly granzyme B, cleave and activate caspases, leading to apoptosis

Triggers and Signals

• DNA damage caused by radiation, chemotherapy, or oxidative stress can activate the intrinsic apoptotic pathway through p53-mediated upregulation of pro-apoptotic Bcl-2 family members
• Growth factor withdrawal can lead to apoptosis by increasing the expression of pro-apoptotic proteins and decreasing the expression of anti-apoptotic proteins
• Endoplasmic reticulum (ER) stress, caused by the accumulation of misfolded proteins or calcium imbalance, can trigger apoptosis through the unfolded protein response (UPR) and activation of caspase-12
• Viral infections can induce apoptosis as a host defense mechanism to limit viral replication and spread
  • Some viruses (adenovirus, Epstein-Barr virus) encode proteins that inhibit apoptosis to promote their survival
• Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells can induce apoptosis in target cells through the perforin/granzyme pathway or by expressing death ligands (FasL, TNF-α)
• Hormones, such as glucocorticoids, can induce apoptosis in certain cell types (thymocytes) by altering the expression of Bcl-2 family proteins
• Oxidative stress, caused by an imbalance between reactive oxygen species (ROS) and antioxidants, can damage cellular components and trigger apoptosis

Stages of Apoptosis

• Initiation: The apoptotic process is triggered by either intrinsic or extrinsic signals, leading to the activation of initiator caspases (caspase-8, -9, or -10)
• Execution: Activated initiator caspases cleave and activate effector caspases (caspase-3, -6, and -7), which in turn cleave various cellular substrates
  • Cleavage of ICAD (inhibitor of caspase-activated DNase) releases CAD, which fragments DNA
  • Cleavage of cytoskeletal proteins (actin, lamin) leads to cell shrinkage and membrane blebbing
  • Cleavage of PARP (poly ADP-ribose polymerase) inhibits DNA repair and conserves cellular energy
• Clearance: Apoptotic cells display "eat-me" signals (phosphatidylserine) on their surface, attracting phagocytic cells for engulfment and removal
  • Apoptotic bodies are formed by the fragmentation of the cell and its contents
  • Phagocytosis of apoptotic cells is an immunologically silent process, preventing inflammation and tissue damage

Cellular Changes During Death

• Cell shrinkage: Cells undergoing apoptosis decrease in size due to the cleavage of cytoskeletal proteins and the loss of cellular fluid
• Chromatin condensation: Nuclear chromatin becomes highly condensed and aggregates along the nuclear membrane
• Nuclear fragmentation: The nucleus breaks down into smaller, discrete fragments as a result of DNA cleavage by caspase-activated DNase (CAD)
• Membrane blebbing: The plasma membrane forms irregular bulges or blebs due to the cleavage of cytoskeletal proteins and the detachment of the membrane from the cytoskeleton
• Apoptotic body formation: The cell and its contents fragment into smaller, membrane-bound vesicles called apoptotic bodies
  • Apoptotic bodies contain organelles, cytosolic components, and nuclear fragments
  • Phosphatidylserine is exposed on the surface of apoptotic bodies, serving as an "eat-me" signal for phagocytic cells
• Mitochondrial changes: Mitochondria undergo morphological and functional alterations, including the loss of membrane potential and the release of pro-apoptotic factors (cytochrome c, AIF, Smac/DIABLO)
• Activation of caspase-dependent and caspase-independent pathways: Caspases cleave various cellular substrates, while other enzymes (AIF, endonuclease G) contribute to apoptotic changes in a caspase-independent manner

Regulation and Control

• The Bcl-2 family of proteins plays a crucial role in regulating the intrinsic apoptotic pathway
  • Pro-apoptotic members (Bax, Bak) promote MOMP and the release of cytochrome c
  • Anti-apoptotic members (Bcl-2, Bcl-xL) inhibit MOMP by sequestering pro-apoptotic proteins
  • BH3-only proteins (Bad, Bid, Bim) act as sensors of cellular stress and promote apoptosis by inhibiting anti-apoptotic proteins or activating pro-apoptotic proteins
• The p53 tumor suppressor protein is a key regulator of apoptosis in response to DNA damage and other cellular stresses
  • p53 transcriptionally upregulates pro-apoptotic genes (Bax, Puma, Noxa) and downregulates anti-apoptotic genes (Bcl-2)
  • p53 can also directly interact with Bcl-2 family proteins to promote MOMP and apoptosis
• Inhibitor of apoptosis proteins (IAPs), such as XIAP, cIAP1, and cIAP2, bind to and inhibit caspases, preventing their activation and the execution of apoptosis
  • IAPs are regulated by Smac/DIABLO, which is released from mitochondria during apoptosis and binds to IAPs, releasing caspases from inhibition
• Post-translational modifications, such as phosphorylation and ubiquitination, can modulate the activity and stability of apoptotic proteins
  • Phosphorylation of Bad by survival kinases (Akt, PKA) promotes its sequestration by 14-3-3 proteins, preventing its pro-apoptotic activity
  • Ubiquitination of caspases and IAPs by E3 ligases can lead to their degradation and the regulation of apoptosis
• MicroRNAs (miRNAs) can regulate apoptosis by targeting the mRNAs of apoptotic proteins for degradation or translational repression
  • miR-15 and miR-16 target Bcl-2 mRNA, reducing its expression and promoting apoptosis
  • miR-34a is induced by p53 and targets the mRNAs of anti-apoptotic proteins, such as Bcl-2 and SIRT1

When Things Go Wrong

• Cancer: Dysregulation of apoptosis is a hallmark of cancer, allowing cells to evade cell death and proliferate uncontrollably
  • Overexpression of anti-apoptotic proteins (Bcl-2, Bcl-xL) or downregulation of pro-apoptotic proteins (Bax, Bak) can promote cancer cell survival
  • Mutations in p53 or other apoptotic regulators can lead to resistance to apoptosis and chemotherapy
• Autoimmune disorders: Defective apoptosis of self-reactive lymphocytes can result in the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)
  • Mutations in the Fas receptor or its ligand (FasL) can lead to the accumulation of self-reactive lymphocytes and autoimmunity
• Neurodegenerative diseases: Excessive apoptosis of neurons can contribute to the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD)
  • Accumulation of misfolded proteins (amyloid-β in AD, α-synuclein in PD, huntingtin in HD) can induce ER stress and apoptosis in neurons
  • Oxidative stress and mitochondrial dysfunction can also trigger apoptosis in neurodegenerative diseases
• Viral infections: Some viruses can modulate apoptosis to promote their replication and spread
  • HIV encodes proteins (Tat, Nef) that can induce apoptosis in uninfected bystander cells, contributing to the depletion of CD4+ T cells
  • Herpesviruses encode anti-apoptotic proteins (vBcl-2, vIAPs) that prevent the death of infected cells, allowing for persistent infection
• Ischemia-reperfusion injury: Apoptosis can occur during the reperfusion of tissues following a period of ischemia, leading to tissue damage and organ dysfunction
  • Ischemia-reperfusion injury is a common complication in myocardial infarction, stroke, and organ transplantation
  • Oxidative stress, calcium overload, and mitochondrial dysfunction during reperfusion can trigger apoptosis in affected tissues

Real-World Applications

• Cancer therapy: Targeting apoptotic pathways is a promising strategy for cancer treatment
  • BH3 mimetics (venetoclax) are small molecules that inhibit anti-apoptotic Bcl-2 proteins, promoting apoptosis in cancer cells
  • TRAIL receptor agonists (dulanermin) can selectively induce apoptosis in cancer cells by activating the extrinsic pathway
  • Inhibitors of IAPs (birinapant, LCL161) can sensitize cancer cells to apoptosis by releasing caspases from inhibition
• Regenerative medicine: Modulating apoptosis can enhance the survival and integration of transplanted cells in regenerative therapies
  • Overexpression of anti-apoptotic proteins (Bcl-2, Bcl-xL) in stem cells can improve their survival and engraftment in harsh environments
  • Inhibition of apoptosis in transplanted pancreatic islets can increase their viability and function in the treatment of diabetes
• Neurodegenerative disease treatment: Preventing excessive apoptosis in neurons is a potential therapeutic approach for neurodegenerative disorders
  • Inhibitors of apoptosis, such as caspase inhibitors (Q-VD-OPh) or ROS scavengers (edaravone), can protect neurons from apoptosis in animal models of AD, PD, and HD
  • Modulating the unfolded protein response (UPR) and reducing ER stress can also mitigate apoptosis in neurodegenerative diseases
• Organ transplantation: Inhibiting apoptosis during organ preservation and transplantation can improve graft survival and function
  • Addition of caspase inhibitors (IDN-6556) or anti-apoptotic proteins (Bcl-xL) to preservation solutions can reduce apoptosis in donor organs
  • Targeting apoptosis in the recipient can also minimize ischemia-reperfusion injury and allograft rejection
• Cardiovascular disease: Preventing apoptosis in cardiomyocytes can limit the extent of myocardial damage following ischemia-reperfusion injury or heart failure
  • Overexpression of anti-apoptotic proteins (Bcl-2, Bcl-xL) in cardiomyocytes can protect against apoptosis and improve cardiac function in animal models
  • Inhibition of pro-apoptotic proteins (Bax, Bak) or caspases can also reduce cardiomyocyte apoptosis and preserve cardiac function