1. Apoptosis is a tightly regulated form of programmed cell death that plays an important role in development, tissue homeostasis, and the immune system. It is characterized by morphological changes including cell shrinkage, chromatin condensation, and formation of apoptotic bodies. 2. The process of apoptosis involves initiator caspases that activate executioner caspases, leading to degradation of nuclear and cytoplasmic components. Mitochondria also play a key role by releasing pro-apoptotic factors. Various proteins regulate apoptosis, including Bcl-2 family members and inhibitors of apoptosis (IAPs). 3. Dysregulation of apoptosis contributes to diseases like cancer, neurodegeneration, and HIV/AIDS. Detection of

1. ADVANCES IN APOPTOTIC PATHWAYS Dr. Pooja Malhotra MODERATOR Dr. Nalini Gupta

2. APOPTOSIS Named after greek designation for “FALLING OFF” Pathway of cell death induced by a tightly regulated intracellular program in which cells destined to die activate enzymes that degrade the cells own nuclear DNA and nuclear and cytoplasmic proteins. Programmed cell death.

3. APOPTOSIS Evolutionarily conserved Occurs in all animals & plants too! Stages and genes conserved from nematodes and flies to humans

4. Physiologic or pathologic Invariably pathologic Physiologic or pathologic Intact released into apoptotic bodies Enzymatic digestion,leak out Cellular contents no frequent Adjacent inflammation Intact,altered structure disrupted Plasma membrane Fragmentation into nuleosome fragments pkynosis ,karyorhexis,karyolysis Nucleus Reduced (shrinkage) Enlarged swelling) Cell size Apoptosis Necrosis Features

6. MORPHOLOGY Cell shrinkage. Chromatin condensation- Most characteristic feature .Formation of Cytoplasmic blebs and apoptotic bodies. Phagocytosis of apoptotic cells or cell bodies by macrophages. PLASMA MEMBRANCES REMAIN INTACT DURING APOPTOSIS UNTIL THE LAST STAGES. Apoptosis doesn’t ilicit inflammation.

9. BIOCHEMICAL FEATURES Protein cleavage. DNA Breakdown. DNA ladders on Agarose gel electrophoresis. DNA smearing on Agarose gel electrophoresis.(necrosis) Phagocytic recognition - Expression of phosphatidyl serine,thrombospondin on outer layer of Plasma membranes.Facilitates recognition by AnnexinV.

12. APOPTOSIS IN PHYSIOLOGIC SITUATIONS Programmed destruction of cells during embryogenesis. Hormone dependent involution in adults. Cell deletion in proliferating cell population. Cell Death of host cells that have outlived their usefulness. Elimination of potentially harmful self reactive lymphocytes. Cell death induced by cytotoxic Tcells as defence against viruses,tumours etc.

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

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

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

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

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

18. APOPTOSIS IN PATHOLOGIC SITUATIONS Cell death by injurious stimuli-radiation,anticancer drugs,heat hypoxia Cell injury in viral diseases-viral hepatitis Pathologic atrophy of parenchymal organs after duct obstruction Cell death in tumours

19. MECHANISMS OF APOPTOSIS Cascade of Molecular Events Iniated by diverse ways Culminate in activation of caspases 2 phases: iniation phase execution phase

20. STAGES OF APOPTOSIS Healthy cell DEATH SIGNAL Commitment to die (reversible) EXECUTION (irreversible) Dead cell (condensed, crosslinked) ENGULFMENT DEGRADATION

21. STAGES OF APOPTOSIS Genetically controlled: Caenorhabditis elegans Soil nematode (worm) Healthy cell Dead cell Committed cell ces2 ces1 ced9 ced3,4 BCL2 Caspases C. Elegans genes == mammalian genes

22. CASPASES : ENEMIES WITHIN Cysteine dependent aspartate specific proteases CYSTEINE ASPARTYL SPECIFIC PROTEASES “ CENTRAL EXECUTIONER OF CELL DEATH”

23. STRUCTURE OF CASPASES Present as inactive zymogens 2 DOMAINS: NH2 Terminal domain(20 KD),LARGE SUBUNIT SMALL SUBUNIT(10 KD) Activation :proteolytic processing between the domains and association of large and small subunits to form heterodimer Two heterodimers associate to form hetrotetramer

25. Absolute requirement for cleavage after ASPARTIC ACID Caspases recognize the Asp residues they cleave within the context of tetrapeptide motifs The preferred tetrapeptide motifs differ among caspases explaining their diversity in biological functions Most proximal (N- terminal)residue –P4 ,Asp-P1 ,cleavage occurs at peptidyl bond distal to targeted Asp.

26. CASPASES 14 CASPASES identified in humans INITIATOR CASPASES/UPSTREAM CASPASES - 8,9,12;have large N terminal prodomains to enable interaction with other proteins to trigger caspase activation EXECUTIONER CASPASES/ DOWNSTREAM CASPASES - 3,7; have short N terminal prodomains Caspases 1,4&5 :not involved in apoptosis but in processing of pro inflammatory cytokines

27. Substrates of effector caspases Protein kinases Signal transduction proteins Cytoskeletal &nuclear matrix proteins Chromatin modifying(poly ADP ribosyl polymerase) DNA repair proteins Inhibitory subunits of endonuclease(CIDE family proteins)

28. Induction of apoptosis : • E xtrinsic or Cell surface death receptor mediated pathway • Mitochondrial-initiated pathway (intrinsic) Endoplasmic reticulum(intrinsic)

30. PROTEIN DOMAINS ASSOCIATED WITH APOPTOSIS REGULATION CASPASE(catalytic domain) DEATH DOMAINS(DD) DEATH EFFECTOR DOMAINS(DED) CASPASE Associated recruitement domains(CARD) BIR (IAP) Bcl-2 homology(BH) Domains

31. CASPASE ACTIVATION MECHANISMS

32. Death Domain proteins Death receptor:signalling and modulators Death receptors detect the presence of extracellular death signals and in response ignite the cells intrinsic apoptosis machinery Belong to TNF receptor gene super family

33. Structure: Cysteine rich extracellular domain Homologous cytoplasmic domain “DEATH DOMAIN (DD)”consisting of compact bundles of six alpha helices

34. DEATH RECEPTORS CD95(Fas or Apo1) - CD95L TNFR1(p55 or CD 120a) – TNF,lymphotoxin DR3(Apo3) - Apo3L(TWEAK) DR4 - TRAIL R1 DR5(Apo2 ) - Apo2L(TRAILR2)

37. APOPTOSIS: control Receptor pathway (physiological): Death receptors: (FAS, TNF-R, etc) FAS ligand TNF Death domains Adaptor proteins Pro-caspase 8 (inactive) Caspase 8 (active) Pro-execution caspase (inactive) Execution caspase (active) Death MITOCHONDRIA

38. ROLE OF CD95-CD95L INTERACTION Peripheral deletion of activated T cells at end of immune response Killing of targets as viral infected cells or cancer cells by cytotoxic T cells&NK cells Killing of inflammatory cells at immune priviliged sites

39. DD NOT ALWAYS INVOLVED IN APOPTOSIS INDUCTION Some non caspase activating DD proteins indirectly regulate apoptosis through effects on NFĸB RIP proteins bind Tradd & activate kinases to cause degradation of IĸB releasing NFĸB to act as transcription factor NFĸB inducible genes include genes for pro inflammatory cytokines ,inhibitors of apoptosis(Bcl-2,IAP members)

40. TNFR1,DR3,DR6 :capable of activating both NFĸB &apoptosis through FADD and Tradd DR4 &DR5: contain only Fadd but not Tradd,hence no activation of NFĸB,no pro inflammatory response& function as effective inducers of apoptosis TRAIL ligand (binds to DR4) is under consideration for clinical use in treatment of cancer

42. ROLE OF MITOCHONDRIA IN APOPTOSIS Mechanisms for release of mitochondrial factor: Damage to mitochondrial membrane Change in membrane potential Altered p53

43. ROLE OF MITOCHONDRIA IN APOPTOSIS CASPASE DEPENDENT PATHWAYS: Smac/Diablo Cytochrome c CASPASE INDEPENDENT PATHWAYS: Apoptosis inducing factor Endonuclease G

46. ENDOPLASMIC RETICULUM AND APOPTOSIS ER stress: Misfolded proteins,Ca 2+ imbalance,loss of oxidizing environment,hypoxia/ischemia Apoptosis (Ca 2+ release,trancription regulated,caspase activation) Caspase 12 knockout mice shown to be resistant to apoptosis induced by amyloid Aß peptide (alzheimers disease)

48. Inhibitors of apoptosis Bcl-2 family of proteins Inhibitors of apoptosis(IAP)

49. Cells are balanced between life and death DAMAGE Physiological death signals DEATH SIGNAL PROAPOPTOTIC PROTEINS ANTIAPOPTOTIC PROTEINS

50. Bcl-2 Family proteins Mitochondrial pathway of apoptosis governed by Bcl-2 family proteins Mediate cross talk between intrinsic and extrinsic pathways Bcl-2 family proteins conserved throughout metazoan evolution Both pro and anti apoptotic proteins are present 20 members of Bcl-2 family gene have been described till date

51. APOPTOSIS: control Intrinsic pathway (damage): Mitochondria Cytochrome c release Pro-caspase 9 cleavage Pro-execution caspase (3) cleavage Caspase (3) cleavage of cellular proteins, Nuclease activation, Etc. Death BAX BAK BOK BCL-Xs BAD BID B IK BIM NIP3 BNIP3 BCL-2 BCL-XL BCL-W MCL1 BFL1 DIVA NR-13 Several viral proteins Pro apoptotic Anti apoptotic

52. Bcl-2 Family proteins Bcl-2 Family proteins divided into 2 groups α helical pore/channel like proteins(Bcl-2, Bcl-XL,Mcl-1,Boo,Bax, Bak, Bid) Have conserved stretches of amino acid sequence homology-BH1,BH2,BH3 Form ion conducting channels in synthetic membranes in vitro

53. Second subset have only BH3 domain in common Include Bad, Bim,Bik, Hrk, APR All are proapoptotic Induce cell death by dimerizing with antiapoptotic Bcl-2 family members and functioning as dominant negative inhibitors of Bcl-2,Bcl- XL Mutations in BH3 domains abolishes their ability to bind Bcl-2 & hence abrogates their ability to induce apoptosis,

57. Cross talk between apoptotic pathways

59. Bcl-2 regulate release of cytochrome c from mitochondria Control release of some caspases(2,3,9)sequestred in some of the cells Release of apoptosis inducing factor(AIF) Release of Smac/Diablo(inhibitor of IAP family proteins)

61. Role of Bcl-2 proteins in disease Bax is increased in apoptosing neurons in alzheimers disease Bax expression is induced in neurons after ischemia reperfusion injury Loss of function mutations noted in human tumors Bcl-2Translocations noted in lymphomas(follicular,HD,etc); overexpression is noted in solid tumoursleading to resistance to chemotherapy and radiation therapy induced apoptosis

62. IAP FAMILY PROTEINS Family of evolutionary conserved apoptotic supressors All members have at least one copy of so called Bir (baculo virus iap repeat)domain required for their antiapoptotic activity 7 members identified so far NAIP,c-IAP1,c-IAP2,Survivin,livin,apollon,XIAP Bind to and inhibit caspase 3,7( BIR domains BIR1&BIR2)&9(BIR 3) XIAP, c-IAP1,c-IAP2 are inhibitors of iniator caspase9 as well as effector caspases 3 &7

64. IAP PROTEINS AND DISEASE Inactivating mutations of NAIP described in Spinal muscular atrophy (a motor neuron degenerative disorder) Overexpression of Survivin seen in many cancers

65. Smac/Diablo and IAP Smac/Diablo binds IAP members- XIAP,Ciap1,Ciap2,survivin and allows apoptosis to occur Smac/Diablo encoded by nuclear genome ,transported into mitochondria and stored in the space between inner & outer membranes Disruption of mitochondrial membrane barrier function leads to activation of these proteins

67. FLIP PROTEINS Belongs to family of proteins containg DED In contrast to other members(FLASH)which enhance apoptosis it supresses caspase 8 activation by Fas & other death receptors FLIP shares sequence homology with procaspase 8 & 10,has a pseudocaspase domain lacking critical regions for protease activity Associates with pro caspase 8 as well as competes with it for binding to Fadd ;thus downregulating apoptosis Decreased expression of FLIP noted in MI

69. NITRIC OXIDE IN APOPTOSIS Possesses both pro &anti apoptotic activity Proapoptotic activity:caspase activation,ability to induce oxidative stress Antiapoptotic activity: FLIP regulation NO negatively regulates Fas CD95 induced apoptosis through inhibition of Ubiquitin Proteasome mediated degradation of FLICE inhibitory protein

70. APOPTOSIS: Role in Disease Neurodegeneration PARKINSON'S DISEASE ALZHEIMER'S DISEASE HUNTINGTON'S DISEASE STROKE

71. STROKE DAMAGED NEURONS MAY COMMIT CELLULAR SUICIDE Recent evidence suggests that neurons die from apoptosis and not necrosis in oxygen deprived brains as occurring during stroke,during perinatal period CASPASE INHIBITORS may be the potential new drugs for stroke

72. G CSF POTENTIAL NEW DRUG FOR STROKE AND NEURODEGENERATIVE DISORDERS Potent hematopietic factor Shows strong antiapoptotic activity in mature neurons G- CSF and its receptor expression increased in ischemia Activates multiple cell survival pathways

73. IS APOPTOSIS THE KEY IN ALZHEIMERS DISEASE? Neurons display characteristic signs of apoptosis such as DNA breaks Apoptosis in alzheimers is induced by Aß protein MUTANT PRESENILINS CONTRIBUTE TO DISEASE BY FURTHER RAISING Aß LEVELS Presenilin mutatins increase neuronal vulnerability to apoptosis

76. 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 defective in apoptotic response High levels of anti-apoptotic proteins or Low levels of pro-apoptotic proteins ===> CANCER

77. APOPTOSIS AND HIV

78. APOPTOSIS AND HIV All T cells express Fas Expression of a HIV gene(nef) causes infected T Cells to express high levels of FasL When infected T cells encounter uninfected one, interaction of FasL with Fas on uninfected cells leads to death by apoptosis

80. DETECTION OF APOPTOSIS IN TUMOUR PARAFFIN SECTIONS Based on morphology Detection of DNA Fragmentation:TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling)method IHC:Detection of cleaved caspase 3: Highly specific marker for apoptosis Can be used as a discriminating criterion to distinguish apoptosis from necrosis

81. IHC DETERMINATION OF CASPASE CLEAVED TARGET PROTEINS Detection of cleaved cytokeratin 18:M30 antibody,precedes TUNEL positivity,represents early event,lost in late stages of c caspase mediatedcleavage of actin fragments recog by FRACTIN FRACTIN stains apoptotic bodies indicating late event PARP(poly ADP ribose polymerase) :one of the earliest nuclear protein cleaved by caspasesinto 89 &24kD fragments ,detects early apoptosis

82. Thank you!