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Apoptosis
Apoptosis
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Apoptosis in health and diseases

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its about pathological basics in apoptosis in health and disease.... pg level

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Apoptosis in health and diseases

  1. 1. APOPTOSIS IN HEALTH & DISEASES MODERATOR : PROF. PARAMESH PRESENTER : DR. MEENU . E.V.
  2. 2. INTRODUCTION
  3. 3. Apoptosis - Definition • A pathway of cell death induced by a tightly regulated suicidal program, in which the cells destined to die activate enzymes that degrade cells own nuclear DNA and nuclear, cytoplasmic proteins. sclero dinesh
  4. 4. Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):239-57 "We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word "apoptosis" (ἁπόπτωσισ) is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees”. mev
  5. 5. Historical aspects • German scientist Carl Vogt - Principle of apoptosis (1842). Mev
  6. 6. • Walther Flemming – Process of programmed cell death (1845). mev
  7. 7. • John Foxton Ross Kerr – Distinguish apoptosis from traumatic cell death (1962). mev
  8. 8. Nobel prize in 2002 – Sydney Brenner , Horvitz, John Buston. ‘identified gene that control apoptosis’ Study done in “Caenorhabditis Elegans”
  9. 9. Significance of apoptosis • During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues [Meier, 2000] • In human body about one lakh cells are produced every second by mitosis and a similar number die by apoptosis (Vaux and Korsmayer ,1999, cell) • 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. ( Karam, Jose A. (2009). Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer. ISBN 978-1-4020-9597-9) • Without apoptosis, human gut can grow up to 12 miles in length • Whole epithelial lining in our body changes every 23 days
  10. 10. sclero dinesh APOPTOSIS NECROSIS NATURAL YES NO EFFECTS BENEFICIAL DETRIMENTAL Physiological or pathological Always pathological Single cells Sheets of cells Energy dependent Energy independent Cell shrinkage Cell swelling Membrane integrity maintained Membrane integrity lost
  11. 11. sclero dinesh APOPTOSIS NECROSIS Role for mitochondria and cytochrome C No role for mitochondria No leak of lysosomal enzymes Leak of lysosomal enzymes Characteristic nuclear changes Nuclei lost Apoptotic bodies form Do not form DNA cleavage No DNA cleavage Activation of specific proteases No activation Regulatable process Not regulated Evolutionarily conserved Not conserved Dead cells ingested by neighboring cells Dead cells ingested by neutrophils and macrophages
  12. 12. WHY APOPTOSIS? To eliminate cells that : • are potentially harmful • have outlived their usefulness / aged • are damaged beyond repair
  13. 13. • Since it is genetically regulated , apoptosis is sometimes referred to as programmed cell death. • Certain forms of necrosis , called “ necroptosis” are also genetically programmed, but by a distinct cell of genes.
  14. 14. MECHANISM OF APOPTOSIS 2 phases of apoptosis 1. Initiation phase - activate the caspases 2 pathways:  Intrinsic / mitochondrial pathway – caspase 9  Extrinsic / receptor mediated pathway - 8 2. Execution phase – caspases causes degradation
  15. 15. INTRINSIC PATHWAY ( MITOCHONDRIAL PATHWAY ) INTRINSIC PATHWAY [ MITOCHONDRIAL PATHWAY ]
  16. 16. PLAYERS 1) Sensors – BAD , BIM , BID 2) Proapoptotic- BAX , BAK 3) Cytochrome C 4) Apaf-1 [ apoptosis activating factor – 1 ] 5) Caspases DEFENDERS 1) Antiapoptotic - BCL 2, BCL XL, 2) IAPS: [ inhibitors of apoptosis
  17. 17. 1.Caspases: • ‘c’ - cysteine protease(an enzyme with cysteine in its active site) ‘aspase’- ability of these enzyme to cleave after aspartic acid residues • More than 10 members • Central executioners of cell death • Depending on order in which they are activated during apoptosis, they are divided into • Initiator caspase - 8, 9 • Executioner caspase -3, 6,7  Presence of cleaved,active caspases is a marker for cells undergoing apoptosis
  18. 18. 2.Bcl-2 proteins: Family of more than 20 members - categorised in 3 groups [ based on proapoptotic and antiapoptotic properties , and presence of BCL-2 homology (BH) domains ] 1) Antiapoptotic - BCL 2, BCL XL • present on outer mito.membrane, cytosol and ER Functn : make outer mitochondrial membrane impermeable and prevent leakage of cytochrome c. Stimulated by : growth factors , survival signals BCL XL - BCL2 related protein, long isoform
  19. 19. 2) Proapoptotic – BAX , BAK Funtn : promote outer mito. Membrane permeability by forming a channel and promote leakage of cytochrome c 3) Sensors – BAD , BIM , BID • Act as sensors of cellular stress and damage • Regulate other two groups BAX – BCL2 associated X protein, BAK- BCL2 antagonist killer 1, BAD- BCL2 antagonist of cell death, BID – BH3 interacting domain death agonist
  20. 20. 3. Cytochrome C – Mitochondrial protein present at inter mitochondrial space – Can activate caspase cascade 4. Apaf-1 [ apoptosis activating factor – 1 ] – Present in cytosol – Forms apoptosome after combining with cytochrome c
  21. 21. 5 . IAPS: [ inhibitors of apoptosis ] – Newly discovered group of anti-apoptotic proteins present in cytosol – 7 members identified-NAIP,cIAP-1,cIAP-2 and survivin – Bind to and inactivate caspases – Survivin is involved in spindle cell formation
  22. 22. INTRINSIC PATHWAY [MITOCHONDRIAL PATHWAY] Withdrawal of Radiation , Toxins, Free radicals survival signals DNA damage / misfolded proteins Loss of antiapoptotic (BCL2, BCLXL ) function ER STRESS activation of sensors of Bcl 2 family activation of proapoptotics- BAX, BAK oligomerization and increase in outer mitochondrial membrane permeability Leakage of cytochrome c leakage of SMAC which bind into cytosol to neutralise IAP’s in cytosol
  23. 23. Contd…… Cytochrome c in cytosol + APAF 1 Form Hexamer ( Apoptosome ) Binds to caspase- 9 ( critical initiator caspase) Enzyme cleaves the adjacent caspase 9 molecules i.e . Autoamplification process Execution phase
  24. 24. EXTRINSIC PATHWAY
  25. 25. Death Receptors Members of TNF receptor family which contain a cytoplasmic domain involved in protein-protein interactions .i.e called Death Domain . 2 types of death receptors are there : • TNFr (tumour necrosis factor receptor ) • FasR (fatty acid synthetase receptor ) The ligand for Fas is called FasL The ligand for TNFr is TNFα Adaptor Proteins also contain death domain a. FADD [ Fas associated death domain ] b. TRADD [ TNF receptor associated death domain]
  26. 26. FAS ligand TNF Death domains Adaptor proteins Pro-caspase 8 (inactive) Caspase 8 (active) Pro-execution caspase (inactive) Execution caspase (active) • Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell • Binding of the complementary deathactivator (FasL and TNF resp ectively) transmits a signal to the cytoplasm • FADD /TRADD attach to death receptors • Binds an inactive form of caspase-8 • M/L procaspase 8 molecules are brought • They cleave and generate active caspase 8
  27. 27. • This pathway can be inhibited by a protein FLIP which binds to pro –caspase 8 and cannot cleave it • Some viruses and normal cells produces FLIP and use this inhibitor to protect themselves from Fas mediated apoptosis FLIP – FLICE inhibitory protein
  28. 28. • Extrinsic and intrinsic pathway involve fundamentally different molecules for their initiation, but their may be an interconnection between them. Eg : in hepatocytes and pancreatic β cells, caspase 8 produced by Fas signaling cleaves and activate sensors of bcl family i.e BID which then stimulate mitochondrial pathway
  29. 29. EXECUTION PHASE • Final phase of apoptosis • Mediated by proteolytic cascade • After initiator caspases ( 2, 8, 9 and 10) are cleaved to generate its active form, the enzymatic death program is set in motion by rapid sequential activation of the executioner caspases – 3,6 • Caspase -3 activates DNase which causes degradation of chromosomal DNA within the nuclei and causes chromatin condensation. • Caspase -3 induces cytoskeletal reorganisation and disintegration of cell into apoptotic bodies.
  30. 30. REMOVAL OF DEAD CELLS Factors by which apoptotic cells attracts phagocytes towards them : • Apoptotic bodies- “bite size”-edible for phagocytes • Phosphatidyl serine “flips” out from inner to outer layer –recognized by macrophages receptors • Some apoptotic bodies express thrombospondin recognized by phagocytes • They are coated by Ab and C1q
  31. 31. 32
  32. 32. MORPHOLOGY OF CELLS IN APOPTOSIS • Cell Shrinkage and dense cytoplasm • Chromatin condensation to periphery and later fragmentation. • Plasma membrane is intact • Formation of cytoplasmic blebs and apoptotic bodies. • Structurally altered so that the apoptotic cell becomes “tasty” for phagocytosis • Phagocytosis of apoptotic bodies by macrophages • Dead cell is rapidly cleared before contents are leaked out • No inflammatory reaction
  33. 33. MORPHOLOGY OF APOPTOSIS Progressive cell shrinkage Chromatin condensation Plasma membrane blebbing Apoptotic bodies Phagocytosis - no inflammation
  34. 34. MORPHOLOGY OF CELLS IN APOPTOSIS
  35. 35. Various methods available for detection of apoptotic cells 1) Light microscopy • In H&E the apoptotic cell appears as round or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin
  36. 36. MICROSCOPY Apoptosis in neutrophils
  37. 37. Councilman bodies- liver tissue
  38. 38. 2) Electron microscopy Defines subcellular changes like • Chromatin condensation • Plasma membrane blebbing
  39. 39. 3) Gel electrophoresis DNA ladder pattern Single cell gel electrophoresis comet assay showing apoptotic cell
  40. 40. 4) TUNEL [ terminal deoxy transferase mediated dUTP nick end labelling ] • Identify DNA breaks in apoptosis Apoptotic cells detected by TUNEL and fluoresce green; while necrotic cells are stained with red-fluorescent propidium iodide
  41. 41. 5) Immunohistochemistry Immunohistochemical detection of apoptotic cells using antibodies against a wide range of substrates most importantly : • Caspase 3 • P53 • Annexin V
  42. 42. APOPTOSIS IN PHYSIOLOGIC CONDITIONS 1) Programmed destruction of cells during embryogenesis including implantation, organogenesis , developmental involution and metamorphosis. Eg : separation of webbed fingers and toes in embryo
  43. 43. Apoptosis in bud formation during which many interdigital cells die. They are stained black by a TUNEL method Incomplete differentiation in two toes due to lack of apoptosis sclero dinesh
  44. 44. 2) Involution of hormone-dependent tissues upon hormone withdrawal… Eg : • Endometrial cell breakdown during menstrual cycle • Ovarian follicular atresia in menopause • Regression of the lactating breast after weaning • Prostatic atrophy after castration
  45. 45. 3) Cell loss in proliferating cell populations Eg : • Immature lymphocytes in bone marrow and thymus that fail to express useful antigen receptors • B lymphocytes in germinal centers • Epithelial cells in intestinal crypts So as to maintain a constant number…
  46. 46. 4) Elimination of potentially harmful self-reactive lymphocytes 5) Death of host cells that have served their useful purpose Eg : - Neutrophils in an a/c inflammatory response - Lymphocytes at the end of an immune response In these situations , cells undergo apoptosis because they are deprived of necessary survival signals such as growth factors.
  47. 47. Apoptosis: Role in Disease TOO MUCH: Tissue atrophy TOO LITTLE: Hyperplasia Neurodegeneration Thin skin etc Cancer Athersclerosis etc
  48. 48. APOPTOSIS IN PATHOLOGIC CONDITIONS Pathological condition arise as a result of dyregulation in apoptosis………………. i.e defective apoptosis with increase cell survival OR Increased apoptosis with increased cell death
  49. 49. DYSREGULATED APOPTOSIS DISORDERS WITH DEFECTIVE APOPTOSIS AND INCREASE CELL SURVIVAL Eg : • Mutations of p53 ------------- CANCERS • AUTOIMMUNE DISEASES DISORDERS WITH INCREASED APOPTOSIS AND EXCESS CELL DEATH Eg : • NEURODENEGERATIVE D/S • Ischemic injury • Death of virus infected cells. Eg -AIDS
  50. 50. Apoptosis: Role in Disease Aging Aging --> both too much and too little apoptosis (evidence for both) Too much (accumulated oxidative damage?) ---> tissue degeneration Too little (defective sensors, signals? ---> dysfunctional cells accumulate hyperplasia (precancerous lesions) sclero dinesh
  51. 51. PREGNANCY ASSOCIATED DISEASE AND APOPTOSIS • During pregnancy trophoblast cells from placenta invade the uterine environment inorder to remodel the maternal blood vessels and help establishing and maintain sucessful pregnancy. • Strict control over the cell proliferation and apoptosis is required to achieve this. • In some cases the process can be compromised and excessive apoptosis of trophoblast cells  failure of fully remodel the maternal environment  complication of pregnancy Eg : preeclampsia of pregnancy
  52. 52. AUTOIMMUNE D/S AND APOPTOSIS IN HEALTHY BODY • T and B – lymphocytes are cells of immune system that are responsible for destroying infected or damaged cell in the body. • They mature in thymus, but before they can enter the blood stream they are tested to ensure that they are effective against foreign antigen and are also not reactive against normal healthy cells. • Any ineffective or self reactive T-cells are removed through induction of apoptosis.
  53. 53. AUTOIMMUNE D/S AND APOPTOSIS ….contd IN DISEASE : • Poor regulation of apoptosis in T- lymphocyte results Autoimmune D/s of cytotoxic T lymphocytes. Eg : Behcet’s D/S, Ankylosing Spondylitis, SLE • Poor regulation of apoptosis in B –cell Eg : SLE , Scleroderma , Multiple sclerosis • RHEUMATOID ARTHRITIS : excessive proliferation of synovial cells is thought to be due in part to the resistence of these cells to apoptosis. • AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME [ALPS] : mutation in FAS gene
  54. 54. Neurodegenerative D/S and Apoptosis IN HEALTHY BODY : • During development of central and peripheral nervous system, many neurons undergo apoptosis that coincides with synaptogenesis. • Signals that determine whether or not developing neurons live or die may include competition for a limited supply of target derived neurotrophic factors and activation of receptors for excitatory neurotransmittor Glutamate. • Initial overproduction of neurons followed by death of some is an adaptive process that provide enough neurons to form nerve cell circuits.
  55. 55. Neurodenerative D/S and Apoptosis…contd Metabolic stress [ stroke, aging ] Oxidative stress and free radicals DNA damage Inherited mutations misfolded proteins ER stress APOPTOSIS increase Ca influx
  56. 56. Neurodenerative D/S and Apoptosis…contd IN DISEASE : • apoptosis of hippocampal neuron  Alzheimer’s D/s Over expression of Bcl 2 • apoptosis of midbrain neurons that uses NT’s dopamine  Parkinson’s Disease • apoptosis of neurons in striatum which control body movements  Huntington’s Disease • apoptosis of lower motor neurons  Amyotrophic Lateral Sclerosis • In stroke  activation of glutamate receptors which act as a trigger to stimulate apoptosis
  57. 57. Apoptosis: Role in Disease Cancer • Apoptosis eliminates damaged cells (damage => mutations => cancer) • Apoptosis is regulated by two major genes p53 & Bcl-2. • Tumor suppressor p53 controls senescence and apoptosis responses to damage. • Mutations or overexpression of these genes will result in Cancer. 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 ===> CANCER
  58. 58. Apoptosis: Role in Disease Cancer Virus associated cancer •Several human papilloma viruses (HPV) have been implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53. •Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas – produces a protein similar to Bcl-2 – produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).
  59. 59. • Some B-cell leukemia and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. • Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1. Apoptosis: Role in Disease Cancer
  60. 60. •Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL). •Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells Apoptosis: Role in Disease Cancer
  61. 61. – Cancer cells • Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells. sclero dinesh Fig. 1: induced apoptosis in stomach carcinoma cells Left: Before induction Middle: 24h after induction Right: 48h after induction
  62. 62. • Apoptosis and AIDS Human Immunodeficiency Virus infects CD4+ T cells and HIV Tat protein increases the expression of Fas receptor, resulting in excessive apoptosis of T cells. Hallmark- the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (µl) of blood). sclero dinesh
  63. 63. ANTI-APOPTOTIC THERAPY IN DISEASES • Stimulation of IAP - for the treatment of stroke, spinalcord injuries , multiple sclerosis • Synthetic nonspecific caspase inhibitors – trials going on treatment of myocardial reperfusion injury, RA • Aim of the treatment in neurodegenerative d/s is to block apoptotic triggers and activation of anti - apoptotic pathway ( by neurotrophic factors ) Eg : block amyloid β production  t/t Alzheimer’s d/s block glutamate receptor activation  stroke using neurotrophic factors like vitamin E  t/t alzheimer’s Insulin like Growth factors  t/t ALS
  64. 64. CONCLUSIONS • Apoptosis is regarded as a carefully regulated energy dependent process, characterized by specific morphological and biochemical features in which caspase activation plays a central role. • The importance of understanding the mechanistic machinery of apoptosis is vital because programmed cell death is a component of both health and disease, being initiated by various physiologic and pathologic stimuli. • Moreover, the widespread involvement of apoptosis in the pathophysiology of disease lends itself to therapeutic intervention at many different checkpoints. • Understanding the mechanisms of apoptosis, and other variants of programmed cell death, at the molecular level provides deeper insight into various disease processes and may thus influence therapeutic strategy.
  65. 65. References • Robbins and Cotran,pathologic basis of disease, Kumar et al, 9th edi.,2014 • Henry’s clinical diagnosis and management,21st edi,2007,546 • Review article on apoptosis, Indian Journal of Cancer ,vol 35, No.4, sep- 2007 , 495-516 • Review article on various method available for detection of apoptotic cell, Indian Journal of Cancer, july-sep 2013 , vol 50, issue 3. • www.ncbi.nlm.nih.gov • www.reading.ac.uk
  66. 66. THANK YOU

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