1. APOPTOSIS, NECROPTOSIS
and AUTOPHAGY
It has been demonstrated that apoptosis is crucial for many biological processes. Therefore, I want to start this presentation by explaining what apoptosis is and which biological phenomena are related to cell death caused by apoptosis.
Associate Professor
Dr. Alexey Podcheko
Spring 2015

2. OBJECTIVES 1. Define apoptosis
2. Compare and contrast the morphologic changes seen in apoptosis versus necrosis
3.Outline the 4 phases of apoptosis
4. Describe the intrinsic and extrinsic pathways of apoptosis

3. OBJECTIVES 5. List 4 physiologic examples of apoptosis
6. List one role of apoptosis in the Pathology of the immune system
7. Describe the role of p53 in carcinogenesis 8. Describe the role of apoptosis in neurodegenerative diseases
9. Define Autophagy
10. List 2 diseases where Autophagy play role in pathogenesis

4. Stages of the cellular response to stress and injurious stimuli

5. The phenomenon of apoptosis in cells was first described by Kerr, Wyllie, and Currie in They discovered morphological changes in cells undergoing cell death that lead to membrane blebbing and digestion of the resulting small vesicles by macrophages. The blebbing cells reminded the researchers of leaves that are scattered around a tree in autumn. From that time on, the Greek word ”apoptosis” not only described the falling of leaves in the autumn, but also the process of programmed cell death.

6. APOPTOSIS DEFINITION:
ENERGY DEPENDENT CONTOLLED PROCESS OF PROGRAMMED CELL DEATH.
Apoptosis is a pathway of cell death that is induced by a tightly regulated suicide program in which cells destined to die activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins.

7. ENERGY DEPENDENT CONTOLLED PROCESS OF PROGRAMMED CELL DEATH

8. Though discovered in 1972, it took almost 20 years for apoptosis to become a focus of biomedical research. The exponential growth of apoptosis publications lead to a better understanding of the biochemical and molecular processes underlying the apoptotic pathways. New scientists enter the apoptosis field every day and contribute to the unraveling of the apoptotic cascades. However, a multitude of mysteries, particularly with diseases, remains to be investigated.

9. Apoptosis
Why should I care?

10. Apoptosis is a genetically programmed process for cells to commit suicide under certain circumstances. Since apoptosis plays a crucial role in tissue homeostasis, often as a counterbalance for cell proliferation, the deregulation of apoptosis often leads to malformations and disease. For diseases as varied as cancer, AIDS, and Alzheimer’s disease, apoptosis plays a major role.

11. (maintains normal size of the organ)
(Chronic myelogenous leukemia)
In tissue development and degeneration, apoptosis and cell proliferation go hand in hand. Deregulation or failure of one of these processes can lead to neoplasia or dystrophy, respectively. In many diseases, such as cancer, it is not enough to propose a lack of apoptosis. Normal apoptosis, in combination with enhanced cell proliferation, can lead to the same results as normal cell proliferation with down-regulated apoptosis. Therefore, closer investigation of underlying disease mechanisms is crucial.
(maintains normal size of the organ)

12. Complexity of Apoptotic pathways
This slide illustrates the complexity of apoptotic pathways. Stimuli such as radiation, hormones, or certain chemicals can directly induce apoptosis by disrupting the cellular DNA and triggering apoptotic events. Internal signals can trigger apoptosis, for example, when mitosis is incomplete and the DNA is damaged. p53 plays a major role in this process. Effector cells and soluble ligands, which bind to apoptosis-related receptors, induce apoptosis via protein cascades that involve various caspases and different kinase pathways.

13. Comparative morphologies of Apoptosis and Necrosis
It is important to realize that there are two different types of cell death: necrosis and apoptosis. Necrosis is also called ”accidental cell death,” and can be caused, for example, by a chemical or physical assault to the cell. This event leads to swelling of the cell and finally disruption of the cell membrane. Organelles, such as the mitochondria or the nucleus, remain intact throughout the process, and all of the cell contents are released into the surrounding tissue. Therefore, cellular necrosis is often associated with inflammation. In contrast, apoptosis is a highly regulated process, and is described as ”programmed cell death.” One crucial step during apoptosis is the release of cytochrome c from the mitochondria into the cytoplasm, followed by the breakdown of the mitochondrial membrane potential. Activation of caspases then leads to cleavage of apoptosis-related proteins (such as PARP, which is involved in DNA repair; or cytokeratin 18, which is important for morphological stability of the cell). A later biochemical hallmark is the ”flipping” of phosphatidylserine from the inner side of the cell membrane to the outer side. DNA condensation and degradation mark one of the last steps occurring in apoptotic cells before the membrane starts to bleb and the resulting vesicles are digested by macrophages. Natural apoptosis usually occurs in single, selected cells, whereas necrosis usually involves larger areas.

14. This slide summarizes the apoptotic events, and compares them with necrosis. It clearly shows that apoptosis and necrosis both lead to cell death, but are completely different processes.

15. 1 2 3 4

16. The two major routes to Apoptosis: Intrinsic and Extrinsic
The extrinsic death receptor pathway is initiated by binding of death ligands to their cognate death receptors
and subsequent recruitment of the adaptor protein FADD and caspase-8/-10 into the caspase-activating death-inducing signaling complex [DISC]. In both pathways effector caspases, such as caspase-3, are activated cleaving selected target proteins. The two pathways are interconnected by the Bcl-2 protein Bid that is cleaved by caspase-8/10 resulting in its mitochondrial translocation and subsequent cytochrome c release.

18. The extrinsic (death receptor–initiated) pathway of apoptosis
DISC – Death inducing signaling complex
FADD- Fas Associated Death Domain
APAF1 – apoptosis activating factor 1
BCL2 family:
1. Anti apoptotic: BCL2, BCL-XL, MCL-1
2. Sensors of damage/proapoptotic: Bim, Bid, Bad
3. Pro apoptotic: Bax, Bak

19. The extrinsic (death receptor–initiated) pathway of apoptosis
Click here to see video

20. The intrinsic or mitochondrial pathway
Involves Bcl family of proteins
Both pro and anti apoptotic members
Best known is anti-apoptotic Bcl2
Production of Bcl2 can be induced by Growth Factors (IGF1) deprivation or IR
The intrinsic mitochondrial pathway is triggered in response to various forms of cellular stress, such as DNA damage, which provokes the activation of one or more proapopotic BH3-only proteins. BH3-only proteins act as sensors for various death stimuli and relieve the inhibitory action of antiapoptotic Bcl-2 proteins. The activation of BH3-proteins results in the oligomerization of Bax and Bak, which are thought to form pores in the outer mitochondrial membrane causing the release of cytochrome c. Subsequently, cytosolic cytochrome c binds to the adaptor protein Apaf-1 and initiates formation of the apoptosome and activation of initiator caspase-9.

21. The mitochondrial pathway of Apoptosis
BCL2 antagonists-
Bim, Bid, and Bad
The mitochondrial pathway is the major mechanism of apoptosis in all mammalian cells, and its role in a variety of physiologic and pathologic processes is well established. This pathway of apoptosis is the result of increased mitochondrial permeability and release of pro-apoptotic molecules (death inducers) into the cytoplasm. Mitochondria are remarkable organelles in that they contain proteins such as cytochrome c that are essential for life, but some of the same proteins, when released into the cytoplasm (an indication that the cell is not healthy), initiate the suicide program of apoptosis. The release of these mitochondrial proteins is controlled by a finely orchestrated balance between pro- and anti-apoptotic members of the Bcl family of proteins. This family is named after Bcl-2, which was identified as an oncogene in a B-cell lymphoma and is homologous to the C. elegans protein Ced-9. There are more than 20 members of the Bcl family, and most of them function to regulate apoptosis. Growth factors and other survival signals stimulate production of anti-apoptotic proteins, the main ones being Bcl-2, Bcl-x, and Mcl-1. These proteins normally reside in the cytoplasm and in mitochondrial membranes, where they control mitochondrial permeability and prevent leakage of mitochondrial proteins that have the ability to trigger cell death. When cells are deprived of survival signals or their DNA is damaged, or misfolded proteins induce ER stress, sensors of damage or stress are activated. These sensors are also members of the Bcl family, and they include proteins called Bim, Bid, and Bad that contain a single “Bcl-2 homology domain” (the third of the four such domains present in Bcl-2) and are called “BH3-only proteins.” The sensors in turn activate two critical (proapoptotic) effectors, Bax and Bak, which form oligomers that insert into the mitochondrial membrane and create channels that allow proteins from the inner mitochondrial membrane to leak out into the cytoplasm. BH3-only proteins may also bind to and block the function of Bcl-2 and Bcl-x. At the same time, the synthesis of Bcl-2 and Bcl-x may decline. The net result of Bax-Bak activation coupled with loss of the protective functions of the anti-apoptotic Bcl family members is the release into the cytoplasm of several mitochondrial proteins that can activate the caspase cascade ( Fig. 1-25B ). One of these proteins is cytochrome c, well known for its role in mitochondrial respiration. Once released into the cytosol, cytochrome c binds to a protein called Apaf-1 (apoptosis-activating factor-1, homologous to Ced-4 in C. elegans), which forms a wheel-like hexamer that has been called the apoptosome.[44] This complex is able to bind caspase-9, the critical initiator caspase of the mitochondrial pathway, and the enzyme cleaves adjacent caspase-9 molecules, thus setting up an auto-amplification process. Other mitochondrial proteins, with arcane names like Smac/DIABLO, enter the cytoplasm, where they bind to and neutralize cytoplasmic proteins that function as physiologic inhibitors of apoptosis (called IAPs). The normal function of the IAPs is to block the activation of caspases, including executioners like caspase-3, and keep cells alive. Thus, the neutralization of these IAPs permits the initiation of a caspase cascade.
Smac/DIABLO
(blocks IAPs)

22. The equilibrium between proapoptotic and antiapoptotic members of the Bcl-2 family of proteins reflects binding of antiapoptotic members to themselves (homodimers) or to the proapoptotic proteins Bak and Bax
FIGURE C. The equilibrium between proapoptotic and antiapoptotic members of the Bcl-2 family of proteins reflects binding of antiapoptotic members to themselves (homodimers) or to the proapoptotic proteins Bak and Bax. Activation of p53, for example, increases production of proapoptotic members of the Bcl-2 family (e.g., Bim, Puma; see Table 1-5|, which outcompete Bax and Bak for binding to Bcl-2 and BcI-Xl, freeing Bax and Bak to open the mitochon-drial permeability pore (MPTP). (continued)

23. Opening of the mitochondrial permeability transition pore leads to Apaf-1 activation and triggering the apoptotic cascade.
CytC- cytochrome c
PTP= permeability transition pores
ROS = reactive oxygen species

24. An experiment introduces a “knockout” gene mutation into a cell line
An experiment introduces a “knockout” gene mutation into a cell line. The frequency of shrunken cells with chromatin clumping and cytoplasmic blebbing is increased compared with a cell line without the mutation. Overall survival of the mutant cell line is reduced. Which of the following genes is most likely to be affected by this mutation?
(A) BAX
(B) p53
(C) C-MYC
(D) FAS
(E) BCL-2

25. How to detect or measure apoptotic events in the tissues ?

26. Apaf-1 (apoptosis-activating factor-1)
This slide shows the sequence of events (in chronological order) that usually occur during apoptosis. Apoptosis induction (e.g., caused by TNF-alpha and Actinomycin D) can be transmitted via receptor activation and cytochrome c release from the mitochondria. The mitochondrial potential decreases, which is the point of no return for the cell to undergo apoptosis. The activation of caspases is intensively investigated since major differences are found in different cell types and tissues. Phosphatidylserine exposure on the outer leaflet of the cell membrane is the first evidence of morphological changes, which later lead to cell shrinkage and blebbing. DNA fragmentation is the last step before disintegration of the cell and phagocytosis of its remains.

27. n this experiment, M30 CytoDEATH was used together with the substrates anti-mouse-biotin, streptavidin-POD, and AEC to detect apoptotic cells in a HeLa cell culture. Hematoxilin was used as a counterstain.

28. - + - + Inducer of Apoptosis
Inducer of Apoptosis
This experiment shows PARP cleavage products detected by western-blot analysis of cell extracts. Apoptosis was induced in MCF-7 cells by treatment with anti-apo1 (lane B) or TNF (lane D). Untreated controls are shown in lanes A and C. The anti-PARP antibody is suitable for immunocytochemistry, histochemistry, immunoprecipitation, and western blotting.
Poly (ADP-ribose) polymerase (PARP) is a protein involved in a number of cellular processes- mainly DNA repair and programmed cell death

29. Phosphatidylserine exposure on the outer leaflet of the cell membrane is the first evidence of morphological changes, which later lead to cell shrinkage and blebbing. DNA fragmentation is the last step before disintegration of the cell and phagocytosis of its remains.

30. Phosphatidylserine exposure during apoptosis
Membrane alterations
Another characteristic feature of apoptosis is the exposure of the phospholipid phosphatidylserine (PS) to the outer cell membrane. PS is normally confined to the inner plasma membrane in healthy cells, but is translocated to the outer membrane leaflet in response to proapoptotic stimuli. This flip-flop mechanism is caspase-dependent, although how caspases promote PS externalization remains a mystery. PS exposure is important for the elimination of apoptotic cells, because it represents an 'eat-me' signal for the engulfment by professional phagocytes following binding to a putative PS receptor. This process therefore ensures the early uptake of apoptotic cells with no release of cellular contents and without provoking an inflammatory response [31].
PS exposure is experimentally detected most commonly using annexin-V, a PS-binding protein. Various annexin-V derivatives coupled to different fluorochromes are available, providing versatile possibilities for apoptosis measurement, for instance by multicolor flow cytometry or fluorescence microscopy (see Annexin-V products on page 39-45). The advantages are sensitivity and rapidity; the disadvantage is that necrotic cells are labeled upon rupture of their plasma membrane. Therefore, it important to control the membrane integrity of the PS-positive cells by double-staining with membrane-impermeable DNA dyes such as propidium iodide. In these assays, healthy cells are doubly negative to annexin-V and propidium iodide, whereas cells in the early phases of apoptosis are annexin-V-positive but propidium iodide-negative, and secondary necrotic cells are doubly positive to annexin-V and propidium iodide.
Annexin-V-FITC
“Eat-me” signal for professional phagocytes

31. Phosphatidylserine exposure during apoptosis
Propidium Iodide stains in red only cells with damaged membranes

32. DNA fragmentation is the last step before disintegration of the cell and phagocytosis of its remains.

33. DNA condensation and fragmentation of 50-300
DNA condensation and fragmentation of base pair fragments is one of the earliest stages of apoptosis! Process of fragmentation will continue until fragment size will reach app. 200 base pairs. Why?

34. Components of a nucleosome
DNA Fragmentation
Components of a nucleosome
Nucleosome is a structure that produces the initial organization of free double-stranded DNA into chromatin. Each nucleosome has an octomeric core complex made up of four types of histones, two copies each of H2A, H2B, H3, and H4. Around this core is wound DNA approximately 150 base pairs in length. One H1 histone is located outside the DNA on the surface of each nucleosome. DNA associated with nucleosomes in vivo thus resembles a long string of beads. Nucleosomes are very dynamic structures, with H1 loosening and DNA unwrapping at least once every second to allow other proteins, including transcription factors and enzymes, access to the DNA

35. Nucleosomal Banding Pattern
DNA Fragmentation
Nucleosomal Banding Pattern
What is the molecular basis of this ladder pattern? Nuclear DNA is attached to a histone complex this is of a specific length. DNA and histones form the so-called nucleosomes that are connected by linker DNA. The linker DNA - in contrast to the histone-bound DNA - is easily accessible to Ca2+-dependent endonucleases that are activated during apoptosis. Cleavage of the DNA preferentially takes place in the linker–region, which leads to the generation of nucleosomes and oligonucleosomal fragments, which can be visualized on an agarose gel as distinct bands. The ladder pattern reflects the fact that the cleavage results in DNA fragments that are in multiples of 200 base pairs, which is the length of the DNA portion protected from DNase digestion in the histone complex.

37. During apoptosis, DNase activity not only generates double-stranded, low-molecular-weight DNA fragments (mono- and oligonucleosomes), but also introduces strand breaks (”nicks”) into the high-molecular-weight DNA. These processes can be identified by labeling the free 3'-OH termini with modified nucleotides using enzymatic reactions. There are two different methods: one uses terminal polymerase to incorporate labeled nucleotides at nicked sites of the DNA, the other uses terminal transferase (TdT) to attach labeled nucleotides to all of the 3' OH-ends.
The second method, which is called TUNEL (TdT-mediated dUTP nick-end labeling) is more sensitive, and is the method used in the Roche Applied Science In Situ Cell Death Detection Kits.

38. This slide shows a TUNEL peroxidase reaction applied to a rabbit endometrium tissue section. To intensify the visualization of apoptotic cells, a counterstaining with hematoxilin was performed.

39. TUNEL-labeled cells can also be analyzed by flow cytometry
TUNEL-labeled cells can also be analyzed by flow cytometry. Panel A shows U937 cells without treatment; panel B shows camptothecin- (CAM-) treated U937 cells. As controls the autofluorescence (green) and the negative control without terminal transferase (blue) are shown. Positively stained cells are clearly visible within the CAM-treated U937 sample (red arrow). Flow cytometry allows easy quantitation of cell populations undergoing apoptosis, which is particularly useful when large sample numbers must be processed.

40. Accumulation of cells in sub-G1 phase of cell cycle during apoptosis
G2/M S G1

41. How apoptotic cells removed from tissues?
Phagocytosis is the main mechanism
Signals for phagocytosis are:
Flip of phosphatidylserine from inner to outer leaflet of cell membrane (ligand for macrophages receptors)
Secretion of soluble factors that recruit phagocytes
Production of thrombospondin on cell surface of apoptotic cells
Coating of apoptotic cells with C1q complement

42. Apoptosis
Why should I care?

43. Apopiosis in postimplamation embryos is involved in processes such as eliminating abnormal, misplaced, nonfunctional, or harmful cells, sculpting structures, eliminating unwanted structures, and controlling cell numbers (46).
Formation of the preamniotic cavity has been shown to occur due to the death of the ectodermal cells in the core of the developing embryo. Apoptosis contributes to the formation of vesicles and tubes (e.g., neural tube) when epithelial sheets invaginatc and tissue inside has to be eliminated (46). Neurons and oligoden-drocytes which are overproduced during the development of the nervous system are also eliminated by apoptosis (47).
One example of the role of apoptosis can be seen in the hand plate. Here, cells which develop between the fingers are eliminated through apoptosis (48).

44. Role of Apoptosis in the Hand Plate
One example of the role of apoptosis can be seen in the hand plate. Here, cells which develop between the fingers are eliminated through apoptosis. The digits themselves begin to form as condensations of initial mesenchymal tissue. These condensations arc the primary signs of future digit location. Apoptosis normally proceeds in zones which do not undergo condensation and is confined to strictly determined areas of mesenchyme. Further apoptosis expands into the whole interdigital mesenchymal tissue. Certain embryonic structures are removed during apoptosis, e.g., the Mullerian duct degenerates in males and the Wolffian duct degenerates in females. In both sexes, the pronephric tubes in mammalian embryos also disappear.

45. A 40 y/o male brought to ED post-MVA
A 40 y/o male brought to ED post-MVA. Patient has a history of fever, headache, chills, and pain in RUQ 5 days ago. Histologic exam shows liver section w/disruption of normal hepatic lobule. Small shrunken hepatocytes w/intense eosinophilic cytoplasm, fragmented nuclear chromatin, & cytoplasmic bleb formations are noted. Which process is most likely occurring in the hepatocytes described? A) apoptosis B) Atrophy C) Caseous necrosis D) Coagulation necrosis E) Dysplasia F) Fatty change G) Hetrophagy H)Liquefaction necrosis I) metaplasia

46. CLINICO-PATHOLOGIC CORRELATIONS: APOPTOSIS AND DISEASE
Reduced Apoptosis – Cancer, Autoimmune Diseases (SLE, Rheumatoid Arthritis, etc)
Increased Apoptosis – Neurodegenerative Diseases, Myocardial Infarction, Stroke, Viral diseases (Hepatitis, AIDS)

47. A 55-year-old woman with colon cancer and a healthy 55-year-old woman are participating a study of colon cancer. Malignant cells are obtained from the tumor in the affected patient, and normal colonic epithelial cells are procured from the healthy subject. After both cell types are treated with transforming growth factor-beta, the number of normal cells decreases, whereas the number of tumor cells remains unchanged. The tumor cells most likely express a mutation that inhibits which of the following physiologic processes? A) Apoptosis B) Cell cycle progression C) DNA repair D) Migration E) Necrosis

48. Mechanisms of increased apoptosis:
Growth Factor Deprivation: Lymphocytes that are not stimulated by antigens and cytokines, neurons deprived of nerve growth factor, endometrial glands in post menopause
DNA Damage: Radiation or chemotherapeutic agents induces DNA damage

49. Role of P53 Tumor suppressor protein in apoptosis and cancerogenesis
P53 is a 53kDa protein present mostly in the nuclei of all cells, rapidly accumulates after injury and become phosporylated
Phosphorylation allows to bind to DNA and activate transcription of cell cycle inhibitors or proapoptotic proteins
P53 block transition of cells from G1 to S phase if DNA damage is not repaired
Picture shows accumulation of P53 protein within nuclei of cells

50. P53 mediated apoptosis

51. Role of P53 Tumor suppressor protein in apoptosis and cancerogenesis
If repair is unsuccessful then P53 will induce expression of BAX. BAX acts against bcl2 and causes apoptosis
Hormonally related cancers seem to particularly involve loss of p53 related apoptosis
Chemo/rad work best when p53 is intact as also act through apoptosis

52. Neurodegenerative Diseases and Apoptosis
- Withdrawal of growth factors favors actions of pro-apoptotic members of Bcl family
- For neurons dependent on nerve growth factors the loss of this stimulation can lead to neural apoptosis and degenerative neurologic diseases:
(Alzheimer's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, Spinal muscular atrophy)

53. Example: SOD-1 mutations activate cell death pathways in familial amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS), sometimes called Lou Gehrig's disease, is a rapidly progressive, invariably fatal neurological disease that attacks the nerve cells (neurons) responsible for controlling voluntary muscles. The disease belongs to a group of disorders known as motor neuron diseases, which are characterized by the gradual degeneration and death of motor neurons.
SOD-1 mutations can activate caspase-1 and caspase-3, and might increase free-radical generation, leading to motor neuron apoptosis. The activation of caspase-1 leads to interleukin-1 production, which can induce a local microglial inflammatory response and increase the number of neurons affected.

54. Mechanisms of IR induced cell death

55. Cell injury caused by virus infection
Direct injury:
-Depletion of cellular resources
-Activation of Apoptosis
Immunologically mediated injury:
-Antibodies+complement
-T-lymphocytes/Granzyme

56. Protein Misfolding and unfolded protein response (aka ER-stress)
Proteins which called “Chaperones” play crucial role in the proper folding of newly synthesized proteins
Normally misfolded polypeptides are ubiquitinated and targeted for proteolysis in proteasomes.
Accumulation of misfolded proteins in the ER trigger a number of cellular responses, collectively called the unfolded protein response
If cells are unable to cope with the accumulation of misfolded proteins, the cell activates caspases and induces apoptosis.
Examples: Alzheimer, Huntington, and Parkinson diseases, type 2 diabetes

57. Mechanisms of protein folding and the unfolded protein response

58. Autophagy
Definition: Autophagy is the process in which a cell sequesters and recycles damaged organelles and macromolecules ( eats its own contents)
What can activate Autophagy:
Nutrient Deprivation( low level of a.acids)
Accumulation of long-living proteins
What regulate Autophagy?
MTOR signaling pathway
Atgs genes/proteins
What Mechanism?
1. Atgs proteins create autophagic vacuoles
2. Autophagic vacuoles fuse with lysosomes and content is digested
Induction: Following external/internal stimuli (e.g. nutrient depletion or ischemia)mTOR is inhibited, leading to induction of Autophagy. Key genes in yeast are Atg1and Atg13, for which the mammalian homologues are yet to be identified.
2) Autophagosome formation: Cytosolic proteins and organelles are sequestered by a double membrane vesicle, the origin of which is uncertain, but may arise from the endoplasmic reticulum. Formation of this vesicle is co-ordinated by complexes of Atg proteins, in particular Atg5 and Atg12, that are conjugated enabling the recruitment of LC3 (Atg8). Beclin-1 forms a complex with Atg14.
3) Docking and fusion with the lysosome
4) Breakdown of the autophagic vesicle. The molecular mechanism behind the fusion with the lysosome and subsequent breakdown of the autophagic vesicle are poorly understood, although Lamp-2 is thought to play a key role.

59. Autophagy and Disease
Cancer: This is an area of active investigation, autophagy can both promote cancer growth and act as a defense against cancers.
Neurodegenerative disorders: In Alzheimer disease, formation of autophagosomes is accelerated. In Huntington disease, mutant protein huntingtin impairs autophagy.
Infectious diseases: Many pathogens are degraded by autophagy; these include mycobacteria, Shigella spp., and HSV-1. This is one way by which microbial proteins are digested and delivered to antigen presentation pathways. Macrophage-specific deletion of Atg5 increases susceptibility to tuberculosis
Inflammatory bowel diseases: Studies have linked both Crohn disease and ulcerative colitis to autophagy related genes.

60. Necroptosis
Necroptosis was first recognized as a caspase-independent form of cell death that can be triggered by treatment with TNF only in the presence of a pan-caspase inhibitors
Main executing molecule of the process is called RIPK3 - receptor-interacting protein kinase 3 (RIPK3)
Necroptosis requires that the function of caspase 8 be inhibited or disrupted.
Unlike apoptosis, in which several of the highly immunogenic intracellular proteins are sequestered in the dead cell, necroptosis is a strong trigger of innate and adaptive immune responses

61. Formation of the Necrosome is key point in the induction of Necroptosis
Molecular mechanism   of   TNF-mediated necroptosis:
Cross-linking   of TNFR1  by  TNF   causes  recruitment   of   RIP1  and   RIP3  along  with  
caspase   8.
Inhibition   of   caspase   8,   as  may  occur  in   some  viral   infections,   allows RIP1  and   RIP3  to initiate signals that affect  mitochondrial  generation  of   ATP and ROS. This  is  followed  by events  typical  of  necrosis.
Molecular  mechanism   of   TNF-mediated  necroptosis:
Cross-
linking   of   TNFR1  by  TNF   causes  recruitment   of   RIP1  and   RIP3  along  with  
caspase   8.   Activation  of   the   caspase   leads   to   apoptosis  as  described  in   the  
text.   Inhibition   of   caspase   8,   as  may  occur  in   some  viral   infections,   allows  
RIP1  and   RIP3  to   initiate   signals   that   affect  mitochondrial  generation  of   ATP  
and  ROS. This  is  followed  by events  typical  of  necrosis.  (Adapted  from  Galluzi  
L,  et   al:  Programmed  necrosis  from  molecules to  health  and  disease. Int Rev 
Cell  Molec  Biol  289:1,   2011.)

62. Key Concepts of Necroptosis
Necroptosis resembles necrosis morphologically and apoptosis mechanistically as a form of programmed cell death.
Necroptosis is triggered by ligation of TNFR1, and viral proteins of RNA and DNA viruses.
Necroptosis is caspase-independent but dependent on signaling by the RIP1 and RIP3 complex.
RIP1-RIP3 signaling reduces mitochondrial ATP generation, causes production of ROS, and permeabilizes lysosomal membranes, thereby causing cellular swelling and membrane damage as occurs in necrosis.
Release of cellular contents evokes an inflammatory reaction as in necrosis.

63. Clinical implications of the necroptosis
Activation of Necroptosis will be beneficial to induce strong anti-viral immune response, e.g. for vaccination or for treatment of viral infections
Inhibition of Necroptosis will be beneficial for treatment of ischemia-reperfusion injury (MI, strokes, transplantation of organs)
More details:

64. Mediation of Programmed Cell Death by Apoptosis or Regulated Necrosis
mitochondrial permeability transition
The term “programmed cell death” was widely used synonymously with
“apoptosis” until necrotic cell death was shown to depend on genetically
defined signaling pathways. Although the role of caspase-mediated apop-tosis in diseases has been revealed in detail over the past three decades,
the contribution of regulated necrosis to the pathophysiological basis of
diseases was investigated only recently. Pathways of regulated necrosis
include necroptosis, which is dependent on receptor-interacting protein
kinase 3 (RIPK3), and regulated necrosis mediated by mitochondrial perme-ability transition (MPT), which involves cyclophilin D–dependent opening
of the MPT pore. Although MPT and necroptosis have been shown to rep-resent two distinct pathways, other emerging signaling cascades of regulated
necrosis have been described, and it remains unclear to what extent such
pathways may have overlapping mechanisms. NAD +
denotes nicotinamide
adenine dinucleotide.

65. Necrostatin1, Necrosulfonamide and Cyclosporine are effective in the prevention of the Ischemia-reperfusion injury

66. Pyroptosis Another form of programmed cell death
Accompanied by the release of fever-inducing cytokine IL-1 from ells
Has some biochemical similarities with apoptosis
Pyroptosis occurs in cells infected by microbes (microbes in the cytoplasm of cells)!!!
Involves activation of caspase-1, generation by cell IL-1
IL-1 recruit leukocytes to the site of infection
As is well known, microbial products that enter the cytoplasm of infected cells are recognized by cytoplasmic innate immune receptors and can activate the multiprotein complex called the inflammasome (Chapter 6). The function of the inflammasome is to activate caspase-1, (also known as interleukin-ip converting enzyme) which cleaves a precursor form of IL-1 and releases its biologically active form. IL-1 is a mediator of many aspects of inflammation, including leukocyte recruitment and fever (Chapter 3). Caspase-1 and, more importantly, the closely related caspase-11 also induce death of the cells. Unlike classical apoptosis, this pathway of cell death is characterized by swelling of cells, loss of plasma membrane integrity, and release of inflammatory mediators. Pyroptosis results in the death of some microbes that gain access to the cytosol and promotes the release of inflammasome-gcncratcd IL-1.

67. From : Cell death in the host response to infection Cell Death and Differentiation (2008) 15, 1339–1349; doi: /cdd ; published online 20 June 2008 , K Labbé1 and M Saleh1,2

68. Pathogen-induced host cell death
Pathogen-induced host cell death. Several forms of host cell death have been described during infection. The type of death the cell undergoes depends on the nature of the pathogen, pathogen load and site of infection. Pyroptotic, apoptotic, autophagic or oncotic cells display a distinct set of morphological and biochemical characteristics, some of which are shared. Whereas apoptosis and autophagy do not induce inflammation, cytokine release and escape of cytoplasmic content during pyroptosis or oncosis are highly inflammatory events. Pathogens are depicted as red ovals. During pyroptosis, pathogens (or pathogenic products) in the cytosol are detected by caspase-1-activating inflammasomes. During apoptosis, pathogens are contained within apoptotic bodies and digested in the lysosomes of phagocytes that engulf apoptotic cells. During autophagy, pathogens are surrounded by autophagosomes and delivered to the lysosomes through autophagosome-lysosome fusion. Although apoptosis, pyroptosis and autophagy are generally beneficial to the host, oncosis favors pathogen dissemination
The type of death the cell undergoes depends on the nature of the pathogen, pathogen load and site of infection.
Pyroptotic, apoptotic, autophagic or oncotic cells display a distinct set of morphological and biochemical characteristics
Apoptosis and autophagy do not induce inflammation
Apoptosis, pyroptosis and autophagy are generally beneficial to the host, oncosis favors pathogen dissemination

69. Please, take your pen and answer on the following questions You have 80 seconds to answer each question

70. Q1: A 43-year-old man presents with a scaly, erythematous lesion on the dorsal surface of his left hand. Skin biopsy reveals atypical Keratinocytes and inflammatory cells, as well as numerous scattered apoptotic bodies. Which of the following proteins plays the most important role in mediating programmed cell death in these Keratinocytes?
A. Catalase
B. Cytochrome c
C. Cytokeratins
D. Myeloperoxidase
E. Superoxide dismutase
Corr B
0:39
0:40
0:38
0:42
0:43
0:37
0:41
0:35
0:32
0:31
0:33
0:34
0:44
0:36
0:46
0:54
0:53
0:55
0:56
0:58
0:57
0:52
0:51
0:47
0:30
0:48
0:49
0:50
0:45
0:28
0:09
0:08
0:10
0:11
0:13
0:12
0:07
0:06
0:01
End
0:02
0:03
0:05
0:04
0:14
0:15
0:24
0:23
0:25
0:26
0:59
0:27
0:22
0:21
0:17
0:16
0:18
0:19
0:20
0:29
1:00
1:41
1:40
1:42
1:43
1:45
1:44
1:39
1:38
1:33
1:32
1:34
1:35
1:37
1:36
1:46
1:47
1:56
1:55
1:57
1:58
2:00
1:59
1:54
1:53
1:49
1:48
1:50
1:51
1:52
1:31
1:30
1:10
1:09
1:11
1:12
1:14
1:13
1:08
1:07
1:03
1:02
1:04
1:05
1:06
1:15
1:16
1:25
1:24
1:26
1:27
1:29
1:28
1:23
1:22
1:18
1:17
1:19
1:20
1:21
1:01

71. Q2: You observe a slide containing numerous epithelial cells
Q2: You observe a slide containing numerous epithelial cells. A colleague looks at the unstained cells under the light microscope and says that most of the cells are undergoing apoptosis. You look at the slide in the microscope and agree. What alterations in the cellular structure did you observe in the light microscope that led to the conclusion that the cells were undergoing apoptosis? A   Plasma membrane was intact  
B   Mitochondria were absent  
C   Plasma membrane showed blebbing  
D   Nucleus was absent  
E   Polysomes were associated with the endoplasmic reticulum 
Corr C
0:39
0:40
0:38
0:42
0:43
0:37
0:41
0:35
0:32
0:31
0:33
0:34
0:44
0:36
0:46
0:54
0:53
0:55
0:56
0:58
0:57
0:52
0:51
0:47
0:30
0:48
0:49
0:50
0:45
0:28
0:09
0:08
0:10
0:11
0:13
0:12
0:07
0:06
0:01
End
0:02
0:03
0:05
0:04
0:14
0:15
0:24
0:23
0:25
0:26
0:59
0:27
0:22
0:21
0:17
0:16
0:18
0:19
0:20
0:29
1:00
1:41
1:40
1:42
1:43
1:45
1:44
1:39
1:38
1:33
1:32
1:34
1:35
1:37
1:36
1:46
1:47
1:56
1:55
1:57
1:58
2:00
1:59
1:54
1:53
1:49
1:48
1:50
1:51
1:52
1:31
1:30
1:10
1:09
1:11
1:12
1:14
1:13
1:08
1:07
1:03
1:02
1:04
1:05
1:06
1:15
1:16
1:25
1:24
1:26
1:27
1:29
1:28
1:23
1:22
1:18
1:17
1:19
1:20
1:21
1:01

72. Q3: On day 28 of her menstrual cycle, a 23-year-old woman experiences onset of menstrual bleeding that lasts for 6 days. She has had regular cycles for many years. Which of the following processes is most likely occurring in the endometrium
just before the onset of bleeding?
(A) Apoptosis
(B) Caseous necrosis
(C) Heterophagocytosis
(D) Atrophy
(E) Liquefactive necrosis
Corr A
0:38
0:39
0:37
0:41
0:42
0:36
0:40
0:34
0:31
0:30
0:32
0:33
0:43
0:35
0:45
0:53
0:52
0:54
0:55
0:57
0:56
0:51
0:50
0:46
0:29
0:47
0:48
0:49
0:44
0:27
0:08
0:07
0:09
0:10
0:12
0:11
0:06
0:05
0:01
End
0:02
0:03
0:04
0:13
0:14
0:23
0:22
0:24
0:25
0:58
0:26
0:21
0:20
0:16
0:15
0:17
0:18
0:19
0:28
0:59
1:40
1:39
1:41
1:42
1:44
1:43
1:38
1:37
1:32
1:31
1:33
1:34
1:36
1:35
1:45
1:46
1:55
1:54
1:56
1:57
1:59
1:58
1:53
1:52
1:48
1:47
1:49
1:50
1:51
1:30
1:29
1:09
1:08
1:10
1:11
1:13
1:12
1:07
1:06
1:01
1:00
1:02
1:03
1:05
1:04
1:14
1:15
1:24
1:23
1:25
1:26
1:28
1:27
1:22
1:21
1:17
1:16
1:18
1:19
1:20
2:00

73. Q4: A cell pathologist performs experiments to examine the biochemical and physiological changes that occur in cells undergoing apoptosis. Which of the following characteristics is associated with the first stage of apoptosis of cells? A   DNA ladder formation  
B   Nuclear condensation  
C   Blebbing or "boiling" of the cytoplasmic membrane  
D   Chromatin condensation  
E   Fragmentation of DNA into kilobase fragments
Corr E
0:39
0:40
0:38
0:42
0:43
0:37
0:41
0:35
0:32
0:31
0:33
0:34
0:44
0:36
0:46
0:54
0:53
0:55
0:56
0:58
0:57
0:52
0:51
0:47
0:30
0:48
0:49
0:50
0:45
0:28
0:09
0:08
0:10
0:11
0:13
0:12
0:07
0:06
0:01
End
0:02
0:03
0:05
0:04
0:14
0:15
0:24
0:23
0:25
0:26
0:59
0:27
0:22
0:21
0:17
0:16
0:18
0:19
0:20
0:29
1:00
1:41
1:40
1:42
1:43
1:45
1:44
1:39
1:38
1:33
1:32
1:34
1:35
1:37
1:36
1:46
1:47
1:56
1:55
1:57
1:58
2:00
1:59
1:54
1:53
1:49
1:48
1:50
1:51
1:52
1:31
1:30
1:10
1:09
1:11
1:12
1:14
1:13
1:08
1:07
1:03
1:02
1:04
1:05
1:06
1:15
1:16
1:25
1:24
1:26
1:27
1:29
1:28
1:23
1:22
1:18
1:17
1:19
1:20
1:21
1:01

74. A male newborn is found to have syndactyly of three fingers of his right hand. As the attending resident you are called upon to explain to the child's parents as to why this happened to their child. Which of the following cellular processes is most likely to have failed during development in utero and caused the malformation? A   Apoptosis  
B   Differentiation  
C   Fusion  
D   Fission  
E   Migration  
F   Proliferation V