Premed · Premed · Cell Biology
Lecture 22: Apoptosis and Programmed Cell Death
Cell Biology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the morphological features that distinguish apoptosis from necrosis
- Explain the intrinsic (mitochondrial) pathway of apoptosis and its regulation by Bcl-2 family proteins
- Explain the extrinsic (death receptor) pathway of apoptosis
- Describe the role of caspases in executing apoptosis
- Discuss the physiological importance of apoptosis and its dysregulation in disease
Lecture Content
I. Apoptosis vs. Necrosis
Apoptosis is a form of programmed cell death that proceeds in an orderly, energy-dependent manner. The dying cell shrinks, and its chromatin condenses (pyknosis) and fragments (karyorrhexis). DNA is cleaved into nucleosomal fragments of approximately 180 base pairs, producing a characteristic ladder pattern on gel electrophoresis. The cytoplasm condenses while organelles initially remain intact, and the plasma membrane undergoes blebbing. The cell ultimately breaks apart into membrane-bound apoptotic bodies. Importantly, phosphatidylserine (PS), normally confined to the inner leaflet of the plasma membrane, is exposed on the outer leaflet, serving as an "eat me" signal. Macrophages and neighboring cells rapidly phagocytose the apoptotic bodies, and because the cell contents never leak into the extracellular space, no inflammatory response is triggered.
Necrosis, by contrast, is an uncontrolled form of cell death resulting from acute injury. The cell swells (oncosis), the membrane ruptures, and cellular contents spill into the extracellular space. The released material includes DAMPs (danger-associated molecular patterns), which trigger an inflammatory response.
Beyond these two classical forms, several other forms of regulated cell death have been recognized. Necroptosis is a programmed form of necrosis mediated by the RIPK1-RIPK3-MLKL pathway that results in an inflammatory outcome. Pyroptosis is an inflammatory cell death mediated by inflammasomes and gasdermin pores, playing an important role in innate immunity. Ferroptosis is driven by iron-dependent lipid peroxidation and is inhibited by the enzyme GPX4.
II. Caspases: The Executioners
Caspases are cysteine-aspartate proteases that cleave their substrates after aspartate residues. They are synthesized as inactive zymogens (pro-caspases) and require proteolytic cleavage for activation.
Initiator caspases are activated first and then activate downstream executioner caspases. Caspase-8 is activated by the death receptor (extrinsic) pathway, while caspase-9 is activated by the mitochondrial (intrinsic) pathway. Initiator caspases contain long prodomains with protein-protein interaction motifs (DED or CARD domains) and are activated by induced proximity, meaning they dimerize on activating platforms.
Executioner caspases carry out the actual demolition of the cell. Caspase-3, -6, and -7 cleave hundreds of cellular substrates to dismantle cellular structures and functions. Key substrates include ICAD (inhibitor of caspase-activated DNase), whose cleavage releases CAD to fragment DNA; lamin A, whose cleavage causes nuclear lamina breakdown and nuclear shrinkage; PARP (poly-ADP-ribose polymerase), whose cleavage inactivates DNA repair; and various cytoskeletal proteins such as actin and fodrin, whose cleavage leads to cell shrinkage and blebbing. Executioner caspases also activate scramblase and inactivate flippase, resulting in the exposure of PS on the outer membrane leaflet. An amplification cascade ensures rapid, irreversible commitment: initiator caspases activate executioner caspases, which can in turn activate additional initiator caspases.
III. The Intrinsic (Mitochondrial) Pathway
The intrinsic pathway is activated by intracellular stresses such as DNA damage, growth factor withdrawal, ER stress, hypoxia, and oncogene activation. The central event is mitochondrial outer membrane permeabilization (MOMP), which releases cytochrome c from the intermembrane space into the cytosol. Additional pro-apoptotic factors are also released, including Smac/DIABLO (which inhibits IAPs), AIF, and endonuclease G.
Bcl-2 family proteins are the critical regulators of MOMP, and they share Bcl-2 homology (BH) domains. The family includes three functional groups. Anti-apoptotic members such as Bcl-2, Bcl-xL, and Mcl-1 reside on the outer mitochondrial membrane where they prevent MOMP by sequestering pro-apoptotic members and maintaining membrane integrity. Pro-apoptotic effectors, principally Bax and Bak, oligomerize in the outer membrane upon activation to form pores that cause MOMP. Bax is cytoplasmic until activated, whereas Bak is constitutively present on the outer membrane. BH3-only proteins such as Bid, Bim, Bad, PUMA, and NOXA act as sensors of stress signals. Among these, activators (Bid, Bim) directly activate Bax and Bak, while sensitizers (Bad, NOXA) bind and neutralize anti-apoptotic proteins to free Bax and Bak. Notably, p53 induces transcription of PUMA and NOXA in response to DNA damage.
Once cytochrome c is released, it binds Apaf-1 (apoptotic protease-activating factor 1) in the cytosol. Apaf-1 undergoes a conformational change requiring dATP and oligomerizes into a heptameric wheel structure called the apoptosome. The apoptosome recruits and activates pro-caspase-9 through CARD-CARD interactions, and active caspase-9 then cleaves and activates caspase-3 and caspase-7.
<image>The intrinsic (mitochondrial) apoptosis pathway. Panel A: Bcl-2 family regulation of MOMP — healthy cell (left): Bcl-2 and Bcl-xL on the outer mitochondrial membrane sequester Bax/Bak; stressed cell (right): BH3-only proteins (PUMA, Bim, Bad) are upregulated, they neutralize anti-apoptotic members and directly activate Bax/Bak, which oligomerize to form pores in the outer membrane. Cytochrome c (red dots) escapes into the cytosol. Panel B: Apoptosome assembly — cytochrome c binds Apaf-1 monomers; Apaf-1 oligomerizes into a heptameric wheel (apoptosome); pro-caspase-9 recruited via CARD domains; active caspase-9 cleaves pro-caspase-3 to generate active caspase-3. Panel C: Balance model — the ratio of anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) to pro-apoptotic (Bax, Bak, BH3-only) proteins determines cell fate; tipping the balance toward pro-apoptotic triggers MOMP and death.</image>
IV. The Extrinsic (Death Receptor) Pathway
The extrinsic pathway is activated by extracellular ligands binding death receptors on the cell surface. Death receptors are members of the TNF receptor superfamily and include Fas (CD95), which binds FasL (CD95L); TNFR1, which binds TNF-alpha; and DR4 and DR5 (TRAIL receptors), which bind TRAIL. All death receptors contain a cytoplasmic death domain (DD).
The signaling mechanism, best illustrated by the Fas pathway, proceeds through a defined series of steps. FasL, a trimer, binds Fas and induces receptor trimerization. The clustered death domains recruit the adaptor protein FADD (Fas-associated death domain). FADD in turn recruits pro-caspase-8 through DED (death effector domain) interactions, assembling the DISC (death-inducing signaling complex). Within the DISC, pro-caspase-8 undergoes induced-proximity dimerization and auto-cleavage to generate active caspase-8. In type I cells such as lymphocytes, active caspase-8 directly cleaves and activates caspase-3.
Cross-talk with the intrinsic pathway occurs in type II cells such as hepatocytes. In these cells, caspase-8 cleaves Bid to generate truncated Bid (tBid), which translocates to mitochondria and activates Bax and Bak, triggering MOMP. This connects to the apoptosome pathway through caspase-9 and ultimately caspase-3, amplifying the death signal through the mitochondrial pathway.
Several regulatory mechanisms modulate the extrinsic pathway. c-FLIP is structurally similar to caspase-8 but catalytically inactive; it competes for binding at the DISC and thereby inhibits caspase-8 activation. Decoy receptors (DcR1, DcR2) bind TRAIL but lack functional death domains, acting as molecular sinks.
<image>The extrinsic (death receptor) apoptosis pathway. Panel A: FasL trimer binds Fas receptors, inducing trimerization and recruitment of FADD (via death domain interaction), followed by pro-caspase-8 recruitment (via DED interaction) forming the DISC. Active caspase-8 directly activates caspase-3 (type I cells). Panel B: Type II amplification — caspase-8 cleaves Bid to tBid; tBid translocates to mitochondria and activates Bax/Bak, triggering MOMP and the intrinsic pathway (cytochrome c release -> apoptosome -> caspase-9 -> caspase-3). Panel C: Inhibitory mechanisms — c-FLIP competes with caspase-8 for DISC binding; IAPs (XIAP) directly inhibit caspase-3, -7, -9; Smac/DIABLO released from mitochondria antagonizes IAPs.</image>
V. Inhibitors of Apoptosis (IAPs) and Their Antagonists
IAP proteins, including XIAP, cIAP1, cIAP2, and survivin, contain BIR (baculovirus IAP repeat) domains that directly bind and inhibit caspases. XIAP is the most potent family member, directly inhibiting caspase-3 and -7 through its BIR2 domain and caspase-9 through its BIR3 domain. cIAP1 and cIAP2 function primarily as E3 ubiquitin ligases that regulate NF-kB signaling. Survivin is expressed only in dividing cells, associates with the mitotic spindle, and also contributes to apoptosis inhibition.
Smac/DIABLO, released from mitochondria during MOMP, counteracts IAP-mediated inhibition by binding IAP BIR domains and displacing caspases, thereby relieving the brake on apoptosis.
These regulators have become important therapeutic targets. Smac mimetics are small molecules that mimic the action of Smac by antagonizing IAPs and are currently in clinical trials for cancer. Venetoclax (ABT-199) is a BH3 mimetic that specifically inhibits Bcl-2, restoring apoptotic sensitivity in cancer cells. Venetoclax is FDA-approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML).
VI. Physiological Roles and Disease
Apoptosis plays essential roles throughout normal physiology. During development, apoptosis sculpts the digits by removing interdigital webs, contributes to neural tube closure, and eliminates approximately 50% of neurons produced during brain development. In the immune system, apoptosis mediates clonal deletion of self-reactive T and B cells and drives contraction of the immune response after pathogen clearance through activation-induced cell death (AICD) via the Fas/FasL pathway. In tissue homeostasis, apoptosis balances cell proliferation and death; the intestinal epithelium, for example, turns over every 3 to 5 days. The DNA damage response relies on p53-mediated apoptosis to eliminate cells harboring irreparable damage.
Diseases of too little apoptosis include cancer and autoimmune disease. In cancer, resistance to apoptosis is a recognized hallmark, achieved through mechanisms such as Bcl-2 overexpression, p53 mutation, and IAP overexpression. Bcl-2 was in fact first discovered in follicular lymphoma, where a t(14;18) translocation places the Bcl-2 gene under the control of the IgH enhancer. In autoimmune disease, failure to eliminate self-reactive lymphocytes drives pathology; mutations in Fas or FasL cause autoimmune lymphoproliferative syndrome (ALPS).
Diseases of too much apoptosis include neurodegenerative diseases, where excessive neuronal apoptosis contributes to Alzheimer's, Parkinson's, and ALS. Ischemic injury involves apoptosis in the penumbra zone following stroke or myocardial infarction. In AIDS, CD4+ T cell depletion occurs partly through apoptosis.
