APOPTOSIS In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis !!!

Introduction  APO-TOE-SIS in greek means “ leaves falling off trees in autum”.  Defination: Apoptosis is a peculiar well controlled individual cell death that is caspase mediated and leads to fragmentation of the cell and organelles into numerous small buds, which are then engulfed by macrophages without surrounding inflammation

Phases of Apoptosis 1. Induction Phase:  In this phase a cell receives a combination of various signals by which  cell cycle arrest  Lack of survival signals  Induction of cell death occurs  DNA damage is started

2. Effector Phase:  In this phase the cell becomes irreversibly committed to death.  There occurs decision process between life and death of cell.  This state is also called as PREAPOPTOSIS.

3. Degradation Phase:  Cell shrinkage (condensation of cytoplasm)  Breakdown of mitochondria; release of cytochrome C  Nuclear condensation  Nuclear fragmentation  Cell membrane blebbing  Fragmentation; apoptotic body formation: membrane-bound cellular fragments, which often lack nuclei  Phagocytosis

How Apoptosis differs from Necrosis 1. Apoptosis is intrinsically controlled, necrosis is not 2. Apoptosis is more rapid (12-24 hours) than necrosis 3. Apoptosis is induced by endogenous or exogenous stimuli, necrosis is always induced by exogenous harms 4. Apoptosis is limited to single or few cells at a time, and occurs among healthy cell population, necrosis is usually more extensive & occurs in tissue exposed to injuries 5. Cell cytoplasm shrinks in apoptosis and swells in necrosis. 6. Nucleosomes of apoptotic cells are 180 bp fragments, contrary to the irregular ones in necrosis 7. Apoptosis has no inflammation, while necrosis leads to liberation of pro-inflammatory mediators 8. Apoptosis has no systemic manifestations contrary to most inflammations

Mechanism of Apoptosis I. Four stages of apoptosis have been defined: i. Committment to death by extracellular or intracellular triggers/signals ii. Cell killing (execution) by activation of intracellular proteases (caspases) iii. Engulfment of cell corpse by other cells iv. Degradation of the cell corpse within the lysosomes of phagocytic cells

Stimuli for Apoptotic Cell Death

Death Factors Definition: cytokines that activate an apoptosis program by binding to their specific receptor. Typical examples of death factors are: 1. Fas ligand, 2. TNF (tumor necrosis factor) and 3. TRAIL (TNF-related apoptosis-inducing ligand). - Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme.

Activation of Caspases  Caspases are central initiators and executioners of apoptosis.  The term caspases is derived from Cysteine- dependent ASPartate-specific proteASES.  14 different members of the caspases-family have been described in mammals Initiator caspases include: 2, 8, 9, 10 Execution caspases include: 3, 6, 7

Apoptopic caspases are subcategorised as: 1. Initiator Caspases (Caspase 2, Caspase 8, Caspase-9, Caspase 10) 2. Executioner Caspases (Caspase 3, Caspase 6 and Caspase 7)  Once initiator caspases are activated, they produce a chain reaction, activating several other executioner caspases.  Executioner caspases degrade over 600 cellular components in order to induce the morphological changes for apoptosis.

 Synthesized as inactive zymogens (PROCASPASES)

The caspase cascade can be activated by:  Granzyme B released by cytotoxic T lymphocytes which is known to activate caspase-3 and -7;  Death receptors (like FAS, TRAIL receptors and TNF receptor) which can activate caspase-8 and -10; and  The apoptosome, regulated by cytochrome c and the Bcl-2 family, which activates caspase-9.

Examples of caspase cascade during apoptosis:

 During times of cellular stress, mitochondrial cytochrome c is released into the cytosol.  This molecule binds an adaptor protein (APAF-1), which recruits initiator Caspase-9.  This leads to the formation of a Caspase activating multiprotein complex called the Apoptosome.  Once activated, initiator caspases such as Caspase 9 will cleave and activate other executioner caspases.  This leads to degradation of cellular components for apoptosis. Intrinsic apoptopic pathway:

Intrinsic Pathway

Extrinsic apoptopic pathway  The caspase cascade is also activated by extracellular ligands, via cell surface Death Receptors. This is done by the formation of a multiprotein Death Inducing Signalling Complex (DISC) that recruits and activates a pro-caspase.  For example, the Fas Ligand binds the FasR receptor at the receptor's extracellular surface; this activates the death domains at the cytoplasmic tail of the receptor.  The adaptor protein FADD will recruit by a Death domain-Death domain interaction. The other end of the DED contains domainfor pro-caspase recruitment. This FasR, FADD and pro-Caspase 8 form the Death Inducing Signalling Complex (DISC) where Caspase-8 is activated. This could lead to either downstream activation of the intrinsic pathway by inducing mitochondrial stress, or direct activation of Executioner Caspases (Caspase 3,6 and 7) to degrade cellular components.

Extrinsic Pathway

Intrinsic pathway Mitochondria Mitochondrial pathway

THE ROADS TO RUIN Apoptotic pathways in multicellular organsims