Apoptosis and Diseases

1. Apoptosis and Diseases

2. Concept • Apoptotic process and changes • Key molecules and Major pathways • Techniques to detect apoptosis • Apoptosis-related diseases • Insufficient apoptosis in diseases • Excessive apoptosis in diseases • Coexistence of insufficient and excessive apoptosis in diseases • Principles of treatment

3. What is Apoptosis? • Apoptosis refers to the process in which the dying procedures that have been in advance deposited in cell are triggered by various causes from in vitro and in vivo, and eventually cause cell death. • Programmed cell death（PCD）

4. Stimulatory Factors Inhibitory Factors Physiological: FasL; Pathological: glutamate, free radicals; therapeutics. Physiological: GFs, estrogen, etc; Pathological: virus; chemicals, etc. Initiation Regulation Execution Conserved Phagocytosis Causes and Process of Apoptosis

5. Apoptotic changes---Morphological changes in apoptosis ---Biochemical Changes in Apoptosis

6. Morphological changes in apoptosis Cell membrane Cytoplasm Cell nucleus Apoptotic body Phagocytose condensation margination Normal Cell Apoptotic Cell Budding Apoptotic Bodies Changes of Cell membrane

7. Apoptosis and Necrosis

8. Morphological differences in apoptosis and necrosis

9. Biochemical Changes in Apoptosis • Caspase activation • Endonuclease activation

10. Caspases: (cysteine-containing aspartate-specific proteases) • Most apoptotic proteolytic cleavage results from the action of caspases • Caspases are activated by proteolytic cleavage • Removal of prodomain and linker region • Assembly of the large and small subunits into an active enzyme complex • Two heterodimers interacting via the small subunits to form a tetramer with two catalytic sites • Family members>14

11. Caspase functions and structure

12. Classification of Caspases

13. 半胱天冬酶的特征

14. Caspase-deficient mice Knockout Phenotype Caspase-1 Viable; impaired processing of IL-1; resistant to endotoxic shock. Caspase-2 Viable; excess numbers of female germ cells; oocytes resistant to chemotherapeutic drugs; B lymphoblasts resistant to granzyme B; accelerated death of facial neurons during development and of sympathetic neurons deprived of NGF. Caspase-3 Lethality at 3–5 weeks of age; defective neuronal apoptosis; T cells resistant to antigen-induced death; abnormal apoptotic morphology in dying cells. Caspase-8 Lethality around E12.5; hyperemia and abnormal heart muscle development; MEFs resistant to TNF, Fas and DR3 but sensitive to UV irradiation, etoposide, staurosporine, serum deprivation. Caspase-9 Perinatal lethal; impaired neuronal apoptosis; ES cells, MEFs and thymocytes generally resistant to intrinsic death stimuli such as DNA damage, though resistance depends on cell type. Caspase-11 Viable; impaired processing of caspase-1, IL-1; resistant to endotoxic shock. Caspase-12 Viable; embryonic fibroblasts are resistant to ER stress.

15. Caspases activation • Death receptor pathway: caspase 8 • Mitochondrial pathway: caspase 9 • ER stress pathway: caspase 12 (CAD: caspase-activated deoxyribonulease)

16. Caspase substrates: • ICAD：DNA降解 • PARP：破坏DNA修复 • 破坏细胞骨架：gelsolin, laminA • 灭活凋亡抑制蛋白：Bcl-2 • etc Cleavage of death substrates

17. Role of Endonuclease:degrade DNA H1 Signaling activation Endonuclease Ca2+ Mg2+ Zn2+ 180-200 bp

18. Apoptotic substrates ( DNA-PKCS, DNA protein kinase catalytic subunit; HnRNP, heteronuclear ribonucleoproteins; ICAD,inhibitor caspase activated deoxyribonuclease; FAK,focal adhesion kinase; GAS,growth arrest specific gene-2; GDI, GDP dissociation inhibitor; NuMA,nuclear mitotic apparatus; PAK,p21 activated kinase;PARP, poly(ADP-ribose) polymerase; cPLA2, cytoplasmic phospholipase A2; RFC-140, replication factor C; SAF-A,scaffold attachment factor-A; U1-70kDa, U1-specific 70-kDa protein; )

19. 凋亡性底物

20. Regulators of ApoptosisBcl2 family proteinsIAP (Inhibitors of Apoptosis Proteins)

21. Bcl2 family: killers and protectors Two groups (>15 members) to keep the balance between apoptosis and survival: ---Suppressors of apoptosis: Bcl2, BclXL, BclW, Bag1, Mcl1, A1, etc ---Activators of apoptosis: Bax, Bok, Hrk, Bnip3, Bim, Bik, BclXs, Bik, Blk, Bid, Bak, Bad, etc. Forms heterodimers • On the cytoplasmic face of the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope • In hematopoietic cell, epithelial cell, lymphocyte, nerve cell, and various cancer cells

22. Bcl-2:Regulate the release of pro-apoptotic molecules from mitochondria Structure of Bcl-2 family TM TM: transmembrane region; BH: Bcl-2 homology

23. Bax: • Apoptotic stimuli induce translocation of Bax from cytosol to mitochondria • create pores in the outer membrane of mitochondria of sufficient size to allow cytochrome C to escape

24. IAPs---family members: c-IAP1,c-IAP2,XIAP,NAIP,survivin;---preventing some procaspases activation, or inhibiting caspase activity.

25. Apoptosis pathways and related genes • Death Receptor induced apoptosis • Mitochondria – Integrator of Apoptosis • ER stress pathway • Others

26. Death Factor and Death Receptor Family

27. Fas ( factor associated suicide): • Homologous cytoplasmic domain: death domain (DD) • Interacts with each other through DD • Anti-apoptotic pathway: NF-kB pathway • TNF rarely induces apoptosis unless protein synthesis is inhibited • Decoy receptors Death receptor induced apoptosis

28. Death Receptor Signaling

29. Apoptosis signaling by CD95, TNFR1, and DR3

30. Apoptosis signaling by DR4 and DR5 and its modulation by decoy receptors

31. Three Types of Killing by the Fas and FasL System • Activation-induced suicide of T cells • CTL-mediated killing of target cells • Killing of inflammatory cells in immune privilege sites and killing of CTL by tumor cells

33. Mitochondria – Integrator of Apoptosis

34. Current models of the intracellular pathways leading to trophic factor mediated cell survival in mammalian cells

35. Current models of the intracellular pathways leading to apoptosis induced by withdrawal of trophic factor

36. ER and Apoptosis

37. Cross-talking among Organelles and Molecules in Apoptosis

39. p53 Mediated Apoptosis

40. p53-Inducible Apoptosis Related Genes Scotin: localized to the ER and the nuclear membrane PERP：similarity to PMP-22/gas3 tetraspan membrane protein NOXA: A member of Bcl-2 family BAX KILLERS/DR5 FAS P53AIP1: p53-regulated apoptosis-inducing protein 1, leads to apoptosis via dissipation of mitochondrial△ψm PIDD: A new death domain containing protein PIG: P53 induced genes,related to ROS production IGFBP

41. The Mammalian DNA Damage Checkpoint

42. Four patterns of death: from apoptosis to necrosis Apoptosis is observed almost exclusively when caspases, in particular caspase-3, are activated. Apoptosis-like PCD chromatin condensation less compact without other apoptotic features “caspase-independent apoptosis” Necrosis-like PCD no chromatin condensation with chromatin clustering to speckles. Usually involves specialized caspase-independent signalling pathways. “aborted apoptosis” Accidental necrosis/cell lysis associated with cellular oedema (organelle swelling) and devoid of zeiosis

43. Techniques to detect apoptosis Morphological studies DNA ladder TUNEL Flow cytometry Externalization of Phosphatidylserine Activation of caspases and cleavage of their substrates

44. Ultrastructural feathers of Normal and Apoptotic Cell Induced Apoptosis of Cultured Rat Hepatocytes

45. DNA Ladder Pattern Seen in Diospyrin diethyl ether Induced Apoptotic Cell

46. Fragmented DNA can be labeled byTerminal deoxynucleotidyl transferase (TdT) mediated deoxyUridine Nucleotide (dUTP)-End Labeling (TUNEL)

47. Flowcytometric Analysis of Cellular DNA Content

48. Externalization of Phosphatidylserine

49. Phosphatidylserine on the surface of apoptotic cells[stained with annexin V (green)] Due to: Caspase-3-mediated cleavage and activation of scramblase PKC activation Inactivated amino- phospholipid translocase

50. Activation of Caspase 3 and Cleavage of Its Substrates, PARP and D4-GDI