ASSIGNMENT ON APOPTOSIS Rajiv Gandhi Proudyogiki Vishwavidyalaya Submitted to Rajiv Gandhi Proudyogiki Vishwavidyalaya Supervised by : Submitted by : Dr. Neelam Balekar Simarpeet Kaur Bhatia M.Pharm, PhD M.Pharm (1st SEM) Professor & Vice Principal COP, IPS ACADEMY, INDORE

INTRODUCTION It is a functional death and it is a good mechanism to eliminate wasted, useless, unwanted, or crippled cells. Process of programmed cell death that may occur in multicellular organisms Leads to characteristic changes like cell shrinkage, nuclear fragmentation, chromatin condensation & chromosomal DNA fragmentation Between 50 & 70 billion cells die each day due to apoptosis in the average human adult.

HISTORY German scientist Carl Vogt was first to describe the principle of apoptosis in 1842 In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. Kerr received the Paul Ehrlich & Ludwig Darmastedter Prize on March 14, 2000, for his description of apoptosis. The 2002 Nobel Prize in medicine was awarded to Sydney Brenner, Horvitz and John E. Sulston for their work regarding apoptosis.

Why should a cell commit suicide? Apoptosis is needed for proper development Examples: The resorption of the tadpole tail The formation of the fingers and toes of the fetus The sloughing off of the inner lining of the uterus The formation of the proper connections between neurons in the brain Apoptosis is needed to destroy cells Cells infected with viruses Cells of the immune system Cells with DNA damage Cancer cells

What makes a cell decide to commit suicide? Withdrawal of positive signals examples : growth factors for neurons Interleukin-2 (IL-2) Receipt of negative signals increased levels of oxidants within the cell damage to DNA by oxidants death activators : Tumor necrosis factor alpha (TNF-) Lymphotoxin (TNF-β) Fas ligand (FasL)

NECROSIS Vs APOPTOSIS Wilde, 199

STAGES OF APOPTOSIS Induction of apoptosis related genes, signal transduction

APOPTOSIS: Morphology organelle reduction membrane blebbing & changes cell shrinkage mitochondrial leakage nuclear fragmentation chromatin condensation

membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events

Blebbing & Apoptotic bodies The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by MFs Apoptotic body MF

Mitochondria/Cytochrome C Apoptosis: Pathways “Extrinsic Pathway” Death Ligands Death Receptors Initiator Caspase 8 Effector Caspase 3 PCD “Intrinsic Pathway” DNA damage & p53 Initiator Caspase 9 Mitochondria/Cytochrome C

MAJOR PLAYERS IN APOPTOSIS Caspases Adaptor proteins TNF & TNFR family Bcl2 family

Ligand-induced cell death Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)

Ligand-induced cell death “The death receptors” Ligand-induced trimerization FasL Trail TNF Death Domains Death Effectors Induced proximity of Caspase 8 Activation of Caspase 8

APOPTOSIS: Signaling & Control pathways I Externally driven Externally driven Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Initiator caspases 6, 8, 9,12 mitochondrion Execution caspases 2, 3, 7 Apoptosis events Activation

APOPTOSIS: Signaling & Control pathways II Externally driven Externally driven Inhibitors Apoptotic signals Activators of initiator enzymes p53 Cytochrome C Internally driven Bcl2 Survival factors External Initiator caspases 6, 8, 9,12 Internal Execution caspases 2, 3, 7 Inhibitors of apoptosis Apoptosis events Inhibition

The mitochondrial pathway Growth factor receptors DNA damage Fas Casp8 PI3K p53 Akt Bid casp3 BAD Bid Bax IAPs casp9 Bid Bcl2 Apaf1 ATP casp3 Bax Cyt.C Smac/ DIABLO H2O2 AIF

REGULATION OF APOPTOSIS Stimuli apoptosis selection of targets Apoptosis by conflicting signals that scramble the normal status of cell Apoptotic stimulicytokines, death factors (FasL) DNA breaks  p53 is activated arrest cell cycle or activate self destruction )

MEASUREMENT OF APOPTOSIS: TECHNIQUES BASED ON MORPHOLOGICAL CHANGES Light microscopy Electron microscopy TECHNIQUES BASED ON DNA FRAGMENTATION Measurement of endonuclease activity

MEASUREMENT OF APOPTOSIS: TECHNIQUES BASED ON MEMBRANE ALTERATIONS Measurement of dye exclusion TECHNIQUES BASED ON CYTOPLASMIC CHANGES Changes in intracellular enzyme activity Measurement of calcium influx

Importance of Apoptosis Important in normal physiology / development Development: Immune systems maturation, Morphogenesis, Neural development Adult: Immune privilege, DNA Damage and wound repair. Excess apoptosis Neurodegenerative diseases Deficient apoptosis Cancer Autoimmunity

APOPTOSIS: important in embryogenesis Morphogenesis (eliminates excess cells): Selection (eliminates non-functional cells):

APOPTOSIS: important in embryogenesis Immunity (eliminates dangerous cells): Self antigen recognizing cell Organ size (eliminates excess cells):

APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Apoptosis Late pregnancy, lactation Involution (non-pregnant, non-lactating) Virgin mammary gland - Testosterone Apoptosis Prostate gland

APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Apoptosis Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery) Steroid immunosuppressant: kill lymphocytes by apoptosis Lymphocytes poised to die by apoptosis

APOPTOSIS: important in adults Maintains organ size and function: X Apoptosis + cell division Cells lost by apoptosis are replaced by cell division

APOPTOSIS: Role in Disease TOO MUCH: Tissue atrophy Neurodegeneration, Thin skin etc TOO LITTLE: Hyperplasia Cancer Atherosclerosis etc

APOPTOSIS: Role in Disease Neurodegneration: Neurons are post-mitotic (cannot replace themselves; neuronal stem cell replacement is inefficient) Neuronal death caused by loss of proper connections, loss of proper growth factors (e.g. NGF), and/or damage (especially oxidative damage) Neuronal dysfunction or damage results in loss of synapses or loss of cell bodies (synaptosis is reversible; apopsosis is irreversible) Also plays a major role in Pakinson’s disease, Alzheimer’s disease & Huntington’s disease

APOPTOSIS: Role in Disease Cancer Apoptosis eliminates damaged cells (damage => mutations => cancer Tumor suppressor p53 controls senescence and apoptosis responses to damage Most cancer cells are defective in apoptotic response (damaged, mutant cells survive) High levels of anti-apoptotic proteins or Low levels of pro-apoptotic proteins causes cancer

APOPTOSIS: Role in Disease Aging Aging --> too much and too little apoptosis (evidence for both) If it is too much (accumulated oxidative damage) ---> tissue degeneration If it is too little (defective sensors, signals) ---> dysfunctional cells accumulate

FUTURE PERSPECTIVES The biological roles of newly identified death receptors and ligands need to be studied Need to know whether defects in these ligands and receptors contribute to disease

CONCLUSION an important process of cell death can be initiated extrinsically through death ligands (e.g. TRAIL, FasL) activating initiator caspase 8 through induced proximity. can be initiated intrinsically through DNA damage (via cytochrome c) activating initiator caspase 9 through oligomerization. Initiator caspases 8 and 9 cleave and activate effector caspase 3, which leads to cell death.

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