Cell Death 2022-23 Gamba PDF

CELL DEATH Si ricorda che tutto il materiale prodotto è protetto da diritto d'autore; può essere utilizzato per finalità di studio e di ricerca a uso individuale e non può essere utilizzato per finalità commerciali, per finalità di lucro anche indiretto (per esempio non può essere condiviso su piat...

CELL DEATH Si ricorda che tutto il materiale prodotto è protetto da diritto d'autore; può essere utilizzato per finalità di studio e di ricerca a uso individuale e non può essere utilizzato per finalità commerciali, per finalità di lucro anche indiretto (per esempio non può essere condiviso su piattaforme online a pagamento o comunque su servizi erogati a scopo di lucro o su siti che guadagnano con introiti pubblicitari). E' inoltre vietata la condivisione su qualsiasi social media di materiale coperto da diritto d'autore, salvo l'adozione di licenze Creative Commons. Si richiama l'attenzione degli studenti a un uso consapevole e corretto dei materiali resi disponibili dalla comunità universitaria, nel rispetto delle disposizioni del codice etico di ateneo. Please note that all the material produced is protected by copyright; it can be used for study and research purposes for individual use and cannot be used for commercial purposes, for profit or indirectly (for example it cannot be shared on paid online platforms or in any case on services provided for profit or on sites that earn with advertising revenue). It is also forbidden to share material covered by copyright on any social media, except for the adoption of Creative Commons licenses. Students' attention is drawn to a conscious and correct use of the materials made available by the University community, in compliance with the provisions of the University’s ethic code. Paola Gamba  CELL DEATH PROCESSES: NECROSIS AND APOPTOSIS Necrosis and apoptosis differ in their morphology, mechanisms, and roles in physiology and disease. Whereas necrosis is always a pathologic process, apoptosis serves many normal functions and is not necessarily associated with cell injury. One of the main research fields nowadays is to understand the molecular mechanisms of cell death and to try to manipulate cell viability. For example: - prolong cardiac myocytes survival after coronary occlusion. - induce cancer cell death.  STRESS ADAPTATION REVERSIBLE CELL INJURY IRREVERSIBLE CELL INJURY CELL DEATH CELL RESISTANCE TO STRESS DEPENDS ON: INTENSITY DURATION GRADE OF SUSCEPTIBILITY OF THE CELL IF THE STIMULUS CAN NO LONGER BE TOLERATED CELL DEATH PHYSIOLOGIC CELL DEATH (apoptosis): Labile cells... PATHOLOGIC CELL DEATH (apoptosis and necrosis): CAUSES: HYPOXIA LACK OF NUTRIENTS EXPOSURE TO TOXINS INFECTIONS DENATURATION OF ENZYMES OR INHIBITION OF THEIR ACTIVITY DENATURATION OF STRUCTURAL PROTEINS (pm)  NECROSIS APOPTOSIS Colloid osmotic lysis Programmed cell death Passive process Active process Acute event that is completed in a few Takes hours minutes Up to a certain point it is reversible, it Once started it is irreversible can regress and the cell can recover its functions The cell components are released in the There is no loss of cellular material (no external environment ( inflammation) inflammation) The cell damage depends on the type of noxious stimuli, not on the Specific molecular pathways mediated characteristics of the cell by genetically determined factors * What happens if a cell undergoing apoptosis exhausts its ATP supply? oncosis + secondary necrosis. Galluzzi et al., Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death & Differentiation (2018) 25:486–541 Galluzzi et al., Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death & Differentiation (2018) 25:486–541  NECROSIS = COLLOID OSMOTIC LYSIS Accidental cell death caused by a hostile environment to which the cell can not adapt effectively. Necrotic cells are unable to maintain membrane integrity and their contents leak out (DAMPs = Damage-Associated Molecular Patterns) INFLAMMATION  Irreversible cell damage (up to a certain point it is reversible). Passive process. Several cells. In the necrotic areas the limits between cells disappear. The enzymes that digest the necrotic cell derive from the lysosomes of the dying cells themselves and from the lysosomes of leukocytes that are called in as part of the inflammatory reaction. 1 Nuclear changes: PYCNOSIS: nuclear shrinkage and increased basophilia. The chromatin condenses into a solid, shrunken basophilic mass (along the nuclear membrane). KARYORRHEXIS: the pycnotic nucleus undergoes fragmentation. KARYOLYSIS: the nucleus in the necrotic cell totally disappears. The fragmented nucleus is completely dispersed in the cytoplasm and can be extruded from the cell. PYCNOSIS KARYORRHEXIS Poli-Columbano, KARYOLYSIS Edizioni Minerva Medica 2 cellular swelling plasma membrane alterations (membrane blebs) denaturation and coagulation of cytoplasmic proteins breaking of the organelles dilation of ER swelling and calcification of mitochondria aggregation of cytoskeleton elements intracytoplasmic myelin figures (large, whorled phospholipid masses derived from damaged cell membranes) 3 Cell disintegration 4 ACUTE INFLAMMATION  COAGULATIVE NECROSIS LIQUEFACTIVE NECROSIS CASEOUS NECROSIS FAT NECROSIS FIBRINOID NECROSIS GANGRENOUS NECROSIS COAGULATIVE NECROSIS: It is the most common cell death due to different kind of noxious stimuli (ISCHEMIA and physical, chemical, infectious agents). Ischemia leads to coagulative necrosis of the supplied tissue in all organ except in the brain (The organs commonly affected are the heart, kidney, and spleen) A localized area of coagulative necrosis is called an infarct. The architecture of dead tissues exhibit a firm texture, it is preserved for a span of at least some days. WHY? The injury denatures not only structural proteins but also enzymes and so blocks the proteolysis of the dead cells. As a result, eosinophilic, anucleate cells may persist for days or weeks. preservation of cellular contours for some time Heart of patient with acute myocardial infarction. Necrotic cells show increased eosinophilia in hematoxylin and eosin stains. Why? Because of: 1) loss of cytoplasmic RNA (which binds the blue dye: hematoxylin) 2) accumulation of denaturated cytoplasmic proteins (which binds to the red dye: eosin) Coagulative necrosis. A wedge- shaped kidney infarct (yellow). Normal kidney (left) and necrotic cells in the infarct area (right) showing preserved cellular outlines with loss of nuclei and an inflammatory infiltrate.  LIQUEFACTIVE NECROSIS Hypoxic death of cells within the central nervous system. Focal bacterial infections, because microbes stimulates the accumulation of leukocytes and the release of enzymes from these cells. The necrotic material is creamy yellow (dead leukocytes) =PUS. - AUTOLYSIS (release of lysosomal enzymes). - ETHEROLYSIS (invasion by strong proteolytic cells, e.g. neutrophil granulocytes). Polymorphonuclear granulocytes of the acute inflammatory response release potent hydrolases able to digest dead cells. In case of bacterial infection: rapid cell death and tissue dissolution. The result is an abscess (cavity formed by liquefactive necrosis in a solid tissue). The colliquated tissue is invaded and removed by macrophage cells and replaced by glial and connective cells. Liquefactive necrosis in a cutaneous abscess. Liquefactive necrosis. An infarct in the brain showing dissolution of the tissue. BIOCHEMICAL MECHANISMS OF CELL DEATH INDUCED BY ISCHEMIA Ischemic necrosis of cardiac myocytes. Loss of selective membrane permeability, leading to membrane damage. NECROSIS  Interruption of blood supply decreases delivery of O2 and glucose Anaerobic glycolysis leads to overproduction of lactate, (piruvate reduced to lactate), ↓ intracellular pH, reduced levels of ATP Distorsion of the activities of pumps in the plasma membrane as a result of the lack of ATP and intracellular acidosis skews the ionic balance of the cell. (the lack of ATP makes the Na/K pump inactive and the Na/H pump pH active: Sodium accumulates!) The increase of intracellular Na activates the Na/Ca pump: Calcium accumulates! The increase of intracellular Ca++ activates PLA2 (degradation of membrane phospholipids) and activates proteases (cytoskeleton destruction) Membrane permeability increases  The lack of oxigen impairs mitochondrial electron transport, thereby decreasing ATP synthesis and facilitating ROS production (lipid peroxidation: damage of mitochondrial membrane) Mitocondrial damage promotes the release of cytochrome c (Cyt c) to the cytosol and initiates the apoptotic cascade. The cell dies! THE "NON-RETURN POINT" BETWEEN REVERSIBLE AND IRREVERSIBLE DAMAGE IS PROBABLY REPRESENTED BY THE OPENING OF THE MPTP (MITOCHONDRIAL PERMEABILITY TRANSITION PORE).  Ca2+ ROLE OF CALCIUM IN THE PATHOGENESIS OF CELL DEATH Many cellular functions are regulated by minimal fluctuations of intracellular Ca++ concentration. Intake of Ca++ from a damaged plasma membrane can cause necrosis (activates PLA2 and proteases causing increased membrane permeability).  CASEOUS NECROSIS Foci of tuberculous infection. Mix between coagulative and liquefactive necrosis. Caseous= cheese-like Friable white appearance of the area of necrosis. Amorphous granular debris enclosed within a distinctive inflammatory border. The term “caseous” (cheeselike) is derived from the friable white appearance of the area of necrosis. Caseous necrosis. Tubercolosis of the lung, with a large area of caseous necrosis containing yellow-white and cheesy debris. Ved. Chronic inflammation  FAT NECROSIS (steatonecrosis) It refers to focal areas of fat destruction Acute pancreatitis: Following the necrosis of the acinar cells of the pancreas there is release of pancreatic digestive enzymes, which digest the pancreas itself and the surrounding tissues, including adipocytes: 1) Phospholipases and proteases attack the plasmamembrane of the adipocytes, which then release the stored TG. 2) TG are hydrolysed by pancreatic lipases producing free FA. 3) Free FA bind Ca++ and precipitate as calcium soaps (saponification). These accumulate as amorphous basophilous deposits at the periphery of the irregular islands of necrotic adipocytes. Fat necrosis. The areas of white chalky deposits represent foci of fat necrosis with calcium soap formation (saponification) at sites of lipid breakdown in the mesentery. Steatonecrosis. Pancreatic adipose tissue of a patient with acute pancreatitis. Island of necrotic adipocytes adjacent to the area of ​acute inflammation. Fatty acids precipitate as calcium soaps and accumulate at the periphery of the foci of necrotic adipocytes.  FIBRINOID NECROSIS Fibrinoid necrosis is a special form of necrosis usually seen in chronic inflammatory processes involving blood vessels. Deposits of fibrin, leaked out of vessels, result in a bright pink and amorphous appearance in H&E stains, called “fibrinoid” (fibrin-like) Gangrenous necrosis not a specific pattern of cell death, but the term is commonly used in clinical practice. It is usually applied to a limb, generally the lower leg, that has lost its blood supply and has undergone necrosis (typically coagulative necrosis) involving multiple tissue planes. When bacterial infection is superimposed there is more liquefactive necrosis because of the actions of degradative enzymes in the bacteria and the attracted leukocytes (giving rise to so-called wet gangrene).  APOPTOSIS Programmed cell death. Death by apoptosis is a normal phenomenon that serves to eliminate cells that are no longer needed, and to maintain a steady number of various cell populations in tissues. It is also a pathologic event when diseased cells become damaged beyond repair and are eliminated. Embryogenesis, metamorphosis  PHYSIOLOGIC APOPTOSIS Involution of hormone-dependent tissues (endometrial cell breakdown during the menstrual cycle, ovarian follicular atresia in menopause, the regression of the lactating breast after weaning, prostatic atrophy after castration). Cell loss in proliferating cell populations (epithelial cells in intestinal crypts… so as to maintain a constant number = homeostasis). Elimination of potentially harmful self-reactive lymphocytes to prevent reactions against one’s own tissues. Death of host cells that have served their useful purpose (neutrophils in an acute inflammatory response, and lymphocytes at the end of an immune response). Morphogenesis (cell death in the interdigital spaces, intestinal epithelium…).  PATHOLOGIC APOPTOSIS DNA damage. If repair mechanisms cannot cope with the injury, the cell triggers intrinsic mechanisms that induce apoptosis. Accumulation of misfolded proteins in the ER (ER stress): degenerative diseases of the central nervous system and other organs. Viral infections, in which loss of infected cells is largely due to apoptosis (several viruses have developed mechanisms to inhibit apoptosis). Radiation, chemotherapy Ischemia ROS  WHY APOPTOTIC PROGRAM? Active program (it needs ATP) Expression of certain genes Genetically programmed cell death The APOPTOTIC CELL undergoes morphological and biochemical changes that lead to its fragmentation and favor phagocytosis. The cell shrinks, losing contact with neighboring cells: cell junctions and membrane specializations disappear. The chromatin aggregates and the nucleus itself may break up. The cellular organelles remain intact. Then the cell is fragmented into APOPTOTIC BODIES that expose specific markers on their surface that act as a signal. They are then phagocytized by neighboring cells that completely degrade them. MORPHOLOGIC FEATURES OF APOPTOSIS: Cell shrinkage. The cell is smaller in size, the cytoplasm is dense, and the organelles, although relatively normal, are more tightly packed. Chromatin condensation. This is the most characteristic feature of apoptosis. The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself may break up, producing two or more fragments. Formation of apoptotic bodies. The apoptotic cell undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragment. Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly ingested by phagocytes and degraded by the phagocyte’s lysosomal enzymes.  The cellular material is not dispersed in the surrounding environment. There is no inflammatory reaction! ? In apoptotic cells, PHOSPHATIDYLSERINE (phospholipid present in the internal leaflet of p.m.) is externally exposed and recognized by tissue macrophages. Different steps involved in efficient apoptotic cell clearance. STEP 1: The find-me signals released by apoptotic cells help attract phagocytes to the proximity of the cell undergoing apoptosis. STEP 2: The phagocytes recognize eat-me signals on apoptotic cells (PS). STEP 3: Cytoskeletal rearrangements and internalization of the dying cell. STEP 4: Release of antiinflammatory cytokines such as TGF-β, IL-10. DOI: 10.1084/jem.20101157  MECHANISMS OF APOPTOSIS Two distinct pathways converge on caspase activation: the mitochondrial pathway and the death receptor pathway INTRINSIC ESTRINSIC PATHWAY PATHWAY  EXTRINSIC PATHWAY Death receptors are members of the TNF receptor family that contain a cytoplasmic domain involved in protein-protein interactions that is called the death domain because it is essential for delivering apoptotic signals  INTRINSIC PATHWAY 1) ROS can activate apoptosis by opening the MPTP (MITOCHONDRIAL PERMEABILITY TRANSITION PORE). Consequent release of CytC. 2) Calcium released by ER induces apoptosis: it is taken up by mitochondria leading to the MPTP opening. Consequent release of CytC. CytC + APAF1 + Caspase 9 = APOPTOSOME Activation of Caspase 3 Activation of DNAse  MITOCHONDRIAL PROTEINS (Bcl2 family) They play a key role in the equilibrium death/survival.. PRO-APOPTOTIC ANTI-APOPTOTIC Binding anti-apoptotic Bax e Bak proteins substitutes Bax Bad Bcl-2 Bak Bid Bcl-XL Bim BclX Bik A1 Nox Ku70 Puma Mcl-1 Noxa homodimers heterodimers Equilibrium between anti-apoptotic Active p53 increases the production of homodimers and pro-anti-apoptotic pro-apoptotic proteins (Bim, Bad, Noxa heterodimers ecc.) Pro-apoptotic activating proteins (Bim, Bik…) compete with Bax and Bak for anti-apoptotic binding and Free Bax e Bak open cause the release of Bax and Bak. MPTP thus releasing CytC. CytC + APAF1 + Caspase 9 = APOPTOSOME Activation of Caspase 3 Activation of DNAse which degrades DNA DNA fragmentation assay for apoptosis detection (DNA laddering). Gel electrophoresis of DNA. A: control B: apoptosis C: necrosis  ROLE OF CALCIUM IN THE PATHOGENESIS OF APOPTOTIC CELL DEATH Ca++ released from ER can induce apoptosis. It can be taken up by mitochondria causing MTPT opening and Cytc release. Moreover, Ca++ may activate caspase 12, that is normally bound to ER membrane. Caspase 12 activates caspase 9 (apoptosome) and consequently caspase 3. ER Caspase 12 Caspase 9 + CytC + APAF1 + Ca2+ Caspase 3 = APOPTOSOME DNAse 1) ACD (Accidental Cell Death) = necrosis 2) RCD (Regulated Cell Death) Apoptosis Necroptosis Pyroptosis, etc.  NECROPTOSIS (programmed necrosis or caspase-indipendent programmed cell death) Hybrid between necrosis and apoptosis It starts in a manner similar to the extrinsic form of apoptosis. - Morphologically it resembles necrosis. - Mechanistically it is triggered by genetically programmed cell signaling transduction (similar to extrinsic apoptosis induced by TNFα). PYROPTOSIS A form of apoptosis characterized by the release of the fever-inducing cytokine IL1. Unlike classical apoptosis, this pathway of cell death is characterized by release of inflammatory mediators. FERROPTOSIS Oxidative stress-dependent. Form of regulated cell death that is triggered when excessive intracellular levels of iron or ROS overwhelm the glutathione-dependent antioxidant defenses to cause membrane lipid peroxidation.