Apoptosis (Intrinsic And Extrinsic Pathway with assays)

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1. • In humans, the rate of cell growth and cell death is balanced to maintain the weight of the body. Introduction Mitosis Apoptosis

2. Introduction Of Apoptosis The word "apoptosis" comes from the ancient Greek, meaning the: "falling of petals from a flower" or "of leaves from a tree in autumn" In 1964 Lockshin, study on programmed cell death. The term apoptosis (a-po-toe-sis) was first used in a now-classic paper by Kerr et al 1972 to describe a morphologically distinct form of cell death.

3. Apoptosis is the process of programmed cell death. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Between 50 and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day.

4. Apoptosis definition Apoptosis or programmed cell death (PCD) is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ("cellular suicide"). It is important for the development of multicellular organism (embryonic development) and homeostasis of their tissues (adult).

5. German scientist Carl Vogt was first to describe the principle of apoptosis in 1842. In 1972 Kerr first introduced the term apoptosis in a publication. Kerr received the Paul Ehrlich and Ludwig Darmstaedter Prize on March 14, 2000, for his description of apoptosis. History

6. The 2002 Nobel Prize in Medicine was awarded to Sydney Brenner, Horvitz and John E. Sulston for their work identifying genes that control apoptosis. Sydney Brenner Horvitz John E. Sulston

7. A P O P T O S I S

8. 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

9. Importance of Apoptosis Apoptosis is a beneficial and important phenomenon:   In embryo 1. During embryonic development, help to digit formation.    Lack of apoptosis in humans can lead to webbed fingers called "syndactyly".

10. Morphogenesis (elimination excess cells) Selection (elimination non-functional cells) Apoptosis Important in embryogenesis

11. Organ size Elimination Excess Cells Immunity Elimination dangerous Cells Self antigen Recognizing cell

12. Normal event in development of nervous system

13. Normal cell turn over Tissue homeostasis Induction and maintenance of immune tolerance  Development of the nervous system  Endocrine-dependent tissue atrophy  Elimination of activated, damaged and abnormal cells In adult

14. Mechanism of cell death Apoptosis = “normal” or “programmed” cell death Apoptosis is the physiological cell death which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis “accidental” or “ordinary” cell death   Necrosis is the pathological cell death which occurs when cells are exposed to a serious physical or chemical insult (hypoxia, hyperthermia, ischemia).

15. Necrosis Apoptosis Accidental /Premature cell death and it is not programed. Programmed cell death Energy independent cell death. Energy dependent cell death It occur when Cell are exposed to harmful chemical or physical agents/conditions. Unwanted cell get eliminated by biological process. Necrotic events: cell swell, rupture, affects surrounding cells too. Apoptotic events: cells become more compact, blebbing of membrane, chromatin condensation, DNA Fragmentation, cell shrink, release of apoptotic bodies. They are inflammatory. Phagocytes involved. Two form of cell death

16. Apoptosis VS Necrosis Necrosis Cellular swelling Membranes are broken ATP is depleted Cell lyses, eliciting an inflammatory reaction DNA fragmentation is random, or smeared In vivo, whole areas of the tissue are affected Apoptosis Apoptosis Cellular condensation Membranes remain intact Requires ATP Cell is phagocytosed, no tissue reaction Ladder-like DNA fragmentation In vivo, individual cells appear affected

17. Apoptosis VS Necrosis

18. 1) Cell shrinkage 2) Organelle reduction 3) Mitochondrial leakage 4) Chromatin condensation 5) Nuclear fragmentation 6) Membrane blebbing & changes Apoptosis- Morphological Changes

19. Apoptosis-Biochemical changes 1)Chromosomal DNA cleaved into fragments 2)Change in the plasma membrane – phosphatidylserine In the outer leaflet  3)Loss of electrical potential across the inner membrane of the mitochondria  4)Relocation of cytochrome c from the intermembrane space of the mitochondria to the cytosol Characteristic biochemical changes in cells undergoing apoptosis

20. Regulation Of Apoptosis 1.Anti-apoptotic factors 2.Pro-apoptotic factors  Example- BCI2 family proteins.

21. BCl2 Family Protein Anti-Apoptotic Factor BCl2, BCl -XL Pro-Apoptotic Factor Bax, Bad, Bik, Bim, PUMA, NOXA

22. What are caspases? Types of caspases How caspases activates? Role in apoptosis/importance What happen in the absence of caspases? Evolution Of Caspase over time

23. What are caspases? Caspases (Cysteine dependent aspartate specific protease) family of protease enzymes. Contain cysteine residue in the catalytic site and selectively cleave proteins at a C-terminal of aspartate. More than 12 caspases are identified 1 2 3 4

24. Caspases Caspases Cysteinyl aspartate specific proteases A family of intracellular cysteine proteases that play a pivotal role in the initiation and execution of apoptosis. At least 14 different members of caspases in mammalian cells have been identified All are synthesized as inactive proenzymes (zymogen) with 32-56 kDa

25. Small subunit (10-13 kDa) Large subunit (17-21 kDa) Prodomain (2-25 kDa) Asp-X Asp-X QACXG 32-56 kDa Effector caspases Initiator caspases Short Long Caspase-2 Caspase-8 Caspase-9 Caspase-10 Caspase-12 Caspase-3 Caspase-6 Caspase-7 Active Caspase C N Caspase structure

26. Caspase subgroups To date, ten major caspases have been identified and broadly categorized into: Signaling or Initiator caspases (2, 8, 9, 10) Effector or Executioner caspases (3, 6, 7) Inflammatory caspases (1, 4, 5) The other caspases that have been identified include: Caspases 11, 12, 13, 14 Central role in cascade of apoptotic events is played by caspase 3 (CPP32)

27. TYPES OF CASPASES Initiator caspases • Caspase-2, 8, 9, 10 • Self activating by autocatalysis EFFECTOR/ EXECUTIONERS Caspase-3, 6, 7  Activated by initiator caspases.

28. How caspases activates? • Caspases are synthesized as inactive zymogens /pro-caspases that are only activated following an appropriate stimulus • This post-translational level of control allows rapid and tight regulation of the enzyme. Pro-caspase split into small and large subunits that form a heterodimer and two such dimers assembles to form the active tetramer.

29. Role in apoptosis/importance Extrinsic pathway of apoptosis Granzyme B pathway. Intrinsic pathway of apoptosis

30. What happen in the absence of caspases? Caspase deficiency has been identified as a cause of tumour development. Tumour growth can occur by a combination of factors, including a mutation in a cell cycle gene and in apoptotic proteins such as Caspases. Evolution Of Caspase In animals apoptosis is induced by caspases and in fungi and plants, apoptosis is induced by arginine and lysine-specific caspase like proteases called metacaspases

31. Recent studies on caspases 1. Over activation of caspase-3 can lead to excessive programmed cell death. 2. Over activation of caspase-3 playing role in causing Alzheimer's disease. 3. Inflammatory caspases- caspase-1, 4, 5, 11, 12

32. APOPTOSIS IN C. elegans ( Nematodes) Four genes whose encoded proteins play an essential role in controlling programmed cell death during C. elegans development: ced-3, ced-4, ced-9, and egl-1. The human Bcl-2 protein and worm CED-9 protein are homologous; even though the two proteins are only 23 percent identical in sequence The first mammalian apoptotic gene to be cloned, bcl-2. Bcl-2 gene can block the extensive cell death found in ced-9 mutant worms. Thus both proteins act as regulators that suppress the apoptotic pathway.  

33. INHIBITORS OF APOPTOSIS(IAPs) Inhibitors of Apoptosis Proteins (IAPs) are a class of highly conserved proteins known for the regulation of caspases and immune signaling. Inhibitor of apoptosis proteins (IAPs), also known as BIRCS (BIR domain containing proteins) are a class of proteins characterized by the presence of Baculovirus IAP Repeat (BIR) domain, a Zn²+ ion coordinating protein-protein interaction motif. They are highly conserved from viruses to mammals.

34. • Yeasts and plants undergo a form of programmed cell death (PCD) by caspase homologs known as metacaspases . • Yet no IAP homologs are found in prokaryotes and plants. • In Yeast the known IAP is BIR1p. • There are eight known mammalian IAPS/BIRCS INHIBITORS OF APOPTOSIS(IAPs)

35. • There are eight known mammalian IAPS/BIRCS 1. BIRC1 (neuronal IAP/NAIP), 2. BIRC2 (cellular IAP1/clAP1), 3. BIRC3 (cellular IAP2/clAP2), 4. BIRC4 (X-linked IAP/XIAP), 5. BIRC5 6. BIRC6 7. BIRC7 (Melanoma IAP/ML-IAP)and 8. BIRC8 (IAP-like protein 2). They function by blocking caspase-3, -7, and -9

36. • Induction of apoptosis in Drosophila requires the activity of three closely linked genes, reaper, hid and grim. • proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (diap1) protein. • Gain-of-function mutations in diap1 strongly suppressed reaper hid- and grim-so no apoptosis. Anti- IAPs in Drosophila

37. Apoptosis pathways Extrinsic pathway (death receptor-mediated events) Death trigger signal from extracellular stimulus and they require the receptor for receiving the signal. Intrinsic pathway (mitochondria-mediated events) Internal damage, So they don’t require Receptor.

38. Reason behind extrinsic response • The extrinsic pathway triggers apoptosis in response to external stimuli, namely by ligand binding at ‘death’ receptors on the cell Surface. • These receptors are typically members of the Tumour Necrosis Factor Receptor (TNFR) gene family, such as TNFR1 or FAS. Binding at these receptors leads to receptor molecules grouping up on the cell surface to initiate downstream caspase activation

39. Components: Death Receptors Death Ligands Adaptor Proteins Caspaseses

40. Death Receptors   " Death receptors" that are members of the tumor necrosis factor (TNF) receptor superfamily.   • Death receptors have a cytoplasmic domain of about 80 amino acids called the "death domain".   • This death domain plays a critical role in transmitting the death signal from the cell surface to the intracellular signaling pathways.

41. Receptor Ligand FasR (CD95/APOI)   DR3   DR4 (TRAIL-RI)   DRS (TRAIL-R2)   TNFRI   TNFR2 Fasl.   Apo3L   Apo21   Apo2L   TNF- α   TNF- β The best characterized receptors & ligands corresponding Death receptors include:

42. Adaptor proteins Apoptotic adaptor proteins play a critical role in regulating pro- and anti-apoptotic signalling pathways  1)FADD ( Fas -associated death domain) 2) TRADD (TNF receptor-associated death domain), are recruited to ligand-activated, oligomerized death receptors to mediate apoptotic signalling pathways.

43.  Two theories of the direct initiation of apoptotic mechanisms in mammals have been suggested: the TNF-induced ( tumor necrosis factor) model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family coupled to extrinsic signals. EXTRINSIC PATHWAY

44. Killer lymphocytes Caspase 8 Caspase 3 Death domain FAS receptor Disc Extracellular signal Ligand and receptor binding FADD recruitment Activation of procaspase 8 Disc formation Activation of executioner caspase Apoptosis of target cell Ligand- Cytokines, TNF, FAS Ligand Receptors-Cytokines receptors, TNF receptors etc

45. Extrinsic Pathway of Apoptosis

46. INTRINSIC PATHWAY / •CASPASE-DEPENDENT INTRINSIC PATHWAY/•MITOCHONDIAL APOPTOSIS REASON OF INTRENSIC PATHWAY Intracellular death signal like DNA damage, Biochemical stress, ROS generation, oncogenic stress, Virus attack or Lack of growth factors. Component Bcl-2 family proteins  Cytochrome c Apoptosome Caspaseses

47. Bcl2 family proteins The control & regulation of apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins Anti-apoptotic proteins include Bcl-2, Bcl -x, Bcl -XL, Bel-w Pro-apoptotic proteins include Bax , Bak , Bid, Bad, Bim , Bik The main mechanism of action of the Bcl-2 family of proteins is the regulation of cytochrome c release from the mitochondria via alteration of mitochondrial membrane permeability.

48. BH4 BH3 BH1 BH2 TM Bcl-2 Bax Bid Bik GROUP I GROUP II GROUP III Group I- Bcl-2 and bcl -xl possess anti apoptotic activity Group II and III- Bax and Bid , Bak possess pro apoptotic activity Bcl2 Homology (BH) domains of Bcl-2 Protein

49. Cytochrome c & Apoptosome Cytochrome c is an abundant protein of the mitochondrial inner membrane, and acts as an electron transport intermediate. During apoptotic activation lead to alterations in permeability of the mitochondrial membrane pore proteins and release of cytochrome c. Cytochrome c binds and activates Apaf-I as well as procaspase-9, forming an "apoptosome”.

50. Intrinsic Pathway The stimuli that initiate the intrinsic pathway produce intracellular signals such as radiation (DNA damage), absence of certain growth factors, hormones and cytokines. All of these stimuli cause changes in the mitochondrial outer membrane permeabilization (MOMP) Release of pro-apoptotic proteins such as cytochrome c, Smac/DIABLO, AIF, endonuclease G and CAD from the intermembrane space into the cytosol. Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an "apoptosome Caspase-9 activation, subsequent caspase-3 activation and cell death.

51. Internal damage signal Inhibition of anti apoptotic protein Oligomerization of Bax/Bak Mitochondrial changes/channel formation of bak or bax Cytochrome C release Activation of Apaf-1 Apoptosome formation Caspase 9 activation Caspase 3 activation

52. Apoptosis assay methods Cytomorphological alterations  DNA fragmentation Detection of caspases, cleaved Substrates, regulators and inhibitors Membrane alterations Detection of apoptosis in whole mounts Mitochondrial assays  Apoptosis assays, based on methodology, can be classified into six Major groups: 

53. Cytomorphological Alterations The evaluation of hematoxylin and eosin-stained tissue sections with light microscopy does allow the visualization of apoptotic cells.  This method detects the later events of apoptosis TEM is considered the gold standard to confirm apoptosis: Electron-dense nucleus Nuclear fragmentation Disorganized cytoplasmic organelles Large clear Vacuoles Intact cell membrane Blebs at the cell surface

54. DNA Fragmentation DNA fragmentation occurs in the later phase of apoptosis. TUNEL (Terminal dUTP Nick End- Labeling ) assay quantified The incorporation of deoxyuridine triphosphate ( dUTP ) at single and double stranded DNA breaks in a reaction catalyzed by the template independent enzyme, terminal deoxynucleotidyl transferase ( TdT ). Incorporated dUTP is labeled such that breaks can be quantified either by flowcytometry, fluorescent microscopy, or light

55. Membrane Alterations During the process of apoptosis, one of the earliest events is Externalization of Phosphatidylserine (PS) from the inner to the outer plasma membrane of apoptotic cells. These cells can be demonstrated by bound with Fluorescein isothiocyanate (FITC)- labeled Annexin V and detected with Fluorescent microscopy.  The vital dye propidium iodide (PI) should be used in combination of annexin V that help in distinguish viable, apoptotic & necrotic cell populations at the same time.

56. Mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway. The mitochondrial outer membrane (MOM) collapses during apoptosis, allowing detection with a fluorescent cationic dye. Cytochrome e release from the mitochondria can also be assayed using fluorescence and electron microscopy in living or fixed cells. Apoptotic or anti-apoptotic regulator proteins such as Bax , Bid, and Bcl-2 can also be detected using fluorescence and confocal microscopy Mitochondrial Assays

57. Recent Advancement   BCL2 inhibitors; 1)G3139 is an antisense oligodeoxynucleotide targeting BCL2 mRNA resulting in RNAse H activation. 2)ABT263 is a small molecule mimetic of the BH3 domain of the pro-apoptotic BAD protein that is currently in clinical trial in chronic lymphatic leukaemia.

58. Caspaseinhibitors   1) VX-765 is an orally active, reversible caspase-1 inhibitor that was being developed for the treatment of inflammatory disorders. 2) Emricasan is a novel, irreversible, orally active pan-caspase inhibitor that has been investigated for the treatment of chronic HCV infection and liver transplantation rejection. 3) NCX-1000, a small-molecule inhibitor that selectively inhibits caspase-3, -8 and -9 in the micromolar range, was in phase II clinical trials for the treatment of chronic liver disease. 4) PAC-1, a small molecule that induces both procaspase-3 activation in vitro and apoptosis in several cancer cell lines