Necrosis, Apoptosis, Autophagy PDF

NECROSIS, APOPTOSIS, AUTOPHAGY HOMEOSTASIS AND ADAPTATION HOMEOSTASIS: is the maintenance of a cellular structure or function with small changes within a limited range ADAPTATION: it is the condition that is created when cells undergo physiological stress or pathological stimuli that le...

NECROSIS, APOPTOSIS, AUTOPHAGY HOMEOSTASIS AND ADAPTATION HOMEOSTASIS: is the maintenance of a cellular structure or function with small changes within a limited range ADAPTATION: it is the condition that is created when cells undergo physiological stress or pathological stimuli that leads them to reach a point of equilibrium other than the state of homeostasis while maintaining a condition of "cellular health". ATROPHY: reduction in cell size (e.g. muscle atrophy after fracture or from innervation) HYPERTROPHY: increase in cell size (e.g., smooth muscle hypertrophy of the uterus during pregnancy; cardiac hypertrophy due to obstructed blood flow into the aorta) HYPERPLASIA: increase in the number of cells (e.g. in athletes, training produces an increase in muscle mass; manual work causes chronic mechanical irritation of the epidermis, which then thickens) METAPLASIA: change in cell type (e.g. in emphysema in which the pulmonary cylindrical epithelium becomes stratified pavement; the same situation is generated in the bronchi of smokers) CELL DAMAGE If the limits of an adaptive response are exceeded or if adaptation is somehow not possible, the cell is unable to maintain its "healthy" state and undergoes abnormal changes. REVERSIBLE DAMAGE: Cell structure and function return to their original condition once physiological stress or pathological stimuli cease to exist. Cellular health is restored. IRREVERSIBLE DAMAGE: if the condition of physiological stress or pathological stimulus persists or is severe from the beginning, the cell reaches a point of no return and undergoes irreversible cell damage and cell death (by necrosis or apoptosis).  If the duration of If the duration of exposure to exposure to the the injurious stimulus is injurious stimulus reduced, the cell loses is prolonged over function, but can recover time or if the injurious stimulus is severe from the * beginning, the cell * undergoes death cambiamenti ultrastrutturali= visibili al microscopio elettronico Cellular response to stress and nociceptive stimuli hyperplasia hypertrophy atrophy metaplasia  Causes of Damage That Lead to cell Necrosis There are several reasons for this: Trauma Ischaemia Anoxia Exposure to heat or freezing Action of poisons or toxins Mechanism of cell damage The mechanisms of action by which the various causes of necrosis lead to cell death may be different, but substantially: Attributable to damage to membrane structures or Disruptions to energy metabolism (Krebs cycle and oxidative phosphorylation are particularly vulnerable). Regardless of the trigger, the leading causes of death are: A. Loss of plasma membrane impermeability with loss of the ability to maintain ionic homeostasis (pore-forming toxins, rhabdomyolysis). B. Oxygen Radicals; C. Decreased ATP synthesis (produced by glycolysis, Krebs cycle and oxidative phosphorylation); decreased ATP synthesis are common consequences of ischemic injury or anoxia in general; The stages of necrosis Necrosis is the result of a fairly stereotyped sequence of events, which we can summarize: 1. Bioenergetic crisis: loss of mitochondrial function, most commonly is the first event of necrosis, loss of ATP production 2. Loss of volumetric homeostasis: the drop in ATP makes membrane ion pumps, which run on ATP, unable to maintain intracellular levels of Na+ and K+, causing 1. increased intracellular Na+, 2. Recall of water from the extracellular environment 3. Cell swelling 4. Plasma membrane distention 5. Swelling of organelle 3. Attempt to survive: the cell tries to stay alive by glycolysis, which also produces ATP, and by activating the so-called heat-shock response, which tries to repair damage to cellular proteins. The stages of necrosis 4. Increased cytosolic Ca2+: from the damaged plasma membrane and the endoplasmic reticulum, which is also damaged, Ca2+ diffuses into the cytosol 5. Activation of Ca2+ dependent phospholipases: phospholipases attack membranes, resulting in 1. Destruction of phospholipids 2. Disorganization of membranes 3. Vicious Circle: Phospholipase-Dependent Damage to Membranes CausesUlteriore aumento di Ca2+ 4. Activation of Ca2+ dependent proteases such as CALPAIN that damage structural proteins (cytoskeleton and membrane proteins) 5. Release of lysosomal enzymes that produce rapid acceleration of cell destruction; Lysosomal deoxyribonucleases digest chromatin 6. Protein denaturation (for cellular acidosis) 7. cell death : The sequence of events precipitates until the cell fails to maintain its homeostasis, the plasma membrane ruptures, and the cell contents spill into the tissue  NECROSIS Lysosome rupture is the most serious event: enzymes are released that begin to damage cellular components Dissolution of the nucleus = KARYOLYSIS  Releases MCP-1 and CX3CL1 that recruit macrophages inflammation DAMPs The type of cell death depends not only on the type of stimulus, but also on the intensity of the stimulus and its duration Stimuli of low intensity and prolonged over time give rise to death by apoptosis. Stimuli of strong intensity but limited in time give death by necrosis. HISTOLOGICAL ASPECTS OF CELL NECROSIS At the histological level, the morphological aspects change, depending on: i) kind of tissue ii) cause of death iii) presence of bacterial superinfection of the necrotic area There are 3 main types of necrosis Coagulative necrosis: frequently caused by ischemia. Perdita del nucleus, Initial preservation of shape and cell profiles with maintenance of the tissue architecture. tissue becomes hard and whitish. It is characterized by the denaturation of cytoplasmic proteins. E’ tipica dei tessuti ricchi di proteine come il cuore. Coagulative necrosis (necrosi coagulativa) Preservation of structure Firm Protein denaturation (including enzymes) due to intracellular acidosis Hypoxic tissue death (except brain) This is an example of coagulative necrosis. This is the typical pattern with ischemia and infarction (loss of blood supply and resultant tissue anoxia). Here, there is a wedge-shaped pale area of coagulative necrosis (infarction) in the renal cortex of the kidney. Spleen: coagulative necrosis  What are the morphologic characteristics of coagulative necrosis? Hypereosinophilia Coagulation of cellular proteins Loss of nuclei (pyknosis, karyorrhexis & karyolysis) Colliquative necrosis: Dead tissue undergoes softening and fluidification phenomena resulting from the activation of autolytic processes (Lytic enzymes of necrosed cells) o heterolithic (lytic enzymes from bacteria, macrophages, etc.). Normal tissue gives way to a viscous liquid mass (pus) Very common when there are bacterial infections*. It is typical of the central nervous system. It is also present in the gastro-intestinal mucosa and pancreas, tissues very rich in proteases. An area of colliquative necrosis at a circumscribed point bounded by a capsule is called abscess. *ma si verifica anche in assenza di infezione Cervello-necrosi colliquativa  Coagulative Liquefactive Figure 1-19 Coagulative and liquefactive necrosis. A, Kidney infarct exhibiting coagulative necrosis, with loss of nuclei and clumping of cytoplasm but with preservation of basic outlines of glomerular and tubular architecture. B, A focus of liquefactive necrosis in the kidney caused by fungal infection. The focus is filled with white cells and cellular debris, creating a renal abscess that obliterates the normal architecture. Caseous necrosis The necrotic tissue is soft and cheese-like; Feature of the central area of tubercular granulomas; The friable and soft texture of the necrotic mass is due in part to the lipid component of the mycobacterial wall. "Unstructured" coagulative necrosis: It differs from coagulative necrosis because in it the normal architecture of the tissue appears to have disappeared (absence of cellular details). After phagocytic lysis, a cavity may remain. Figure 1-20 A tuberculous lung with a large area of caseous necrosis. The caseous debris is yellow-white and cheesy. Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 13 October 2009 08:39 AM) © 2007 Elsevier Fat necrosis (steatonecrosi) Not a specific pattern Focal areas of fat digestion Usually via release of lipases from pancreas FFA combine with Ca2+ to produce “soaps” (fat saponification) Tipical in acute pancreatitis Figure 1-21 Foci of fat necrosis with saponification in the mesentery. The areas of white chalky deposits represent calcium soap formation at sites of lipid breakdown. Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 13 October 2009 08:39 AM) © 2007 Elsevier  APOPTOSIS The orderly development and homeostatic maintenance of multicellular organisms not only requires nutrients and adequate growth potential, but is also based on complex mechanisms of cell suicide. apoptosis is «programmed» cell death, used for the elimination of cells that are no longer needed or damaged through the activation of a sequence of coordinated and programmed events carried out by a series of highly conserved proteins from the simplest multicellular organisms to humans. Apoptosis, unlike necrosis, does not trigger inflammation and the resulting apoptotic corpuscles are eliminated by phagocytosis mainly by macrophages recalled by MCP-1 and CX3CL1 Examples of physiological apoptosis (i) - During Embryonic development - mechanism of tissue remodeling and maintenance of cellular homeostasis (e.g. intestines, skin)- - As a mechanism of Immune reactions: apoptosis in CD8 T cell target cells and elimination of self-responsive lymphocytes (negative selection) - aging Examples of physiological apoptosis (ii) Morphogenesis (excess of cells) Immune mechanisms: elimination of self-responding lymphocytes; Examples of physiological apoptosis (iii) Tissue remodeling (useless cells): Apoptosis Mammary gland lactation involution - Testosterone Apoptosis Prostate  APOPTOSIS INDUCERS DNA DAMAGE: radiation, cytotoxic drugs used to treat cancer, oxygen radicals. ENDOGENOUS If the repair mechanisms cannot cope with the damage, the apoptotic process is triggered. ACCUMULATION OF MISFOLDED PROTEINS: they can be formed due to mutations in the genes that encode them or as a product of damage produced by free radicals. LACK OF GROWTH FACTORS EXOGENOUS DEATH RECEPTOR LIGANDS (FAS-L and TNF-a) CHARACTERISTICS OF APOPTOSIS Apoptosis is guided and tightly controlled by a set of genetic and biochemical programs that, once activated, produce a stereotyped series of cellular alterations: Cytoplasmic wrinkle; detachment from the matrix; mitochondrial swelling (up to a certain point the mitochondria function: energy is needed for apoptosis); Chromatin aggregates in the periphery of the nucleus (under the membrane) DNA is degraded in a regular manner (laddering) with cuts at the nucleosomal level, as well as nuclear proteins; the nucleus condenses and fragments (pycnosis and karyorexis) Formation of vesicles (blebs) resulting from fragmentation of the cytoplasm that detach from the cell body giving apoptotic bodies; Loss of normal plasma membrane lipid asymmetry with exposure on the cell surface of PS, which is essential for macrophage binding (eat-me signal); The different stages of apoptosis: the arrow indicates the apoptotic body Membrane blebbing DNA fragmentation: 180 bp fragments are formed Apoptosis is caused by the activation of enzymes: CASPASE Cysteine Proteases: cut proteins at the level of aspartic acid residues (hence C-ASP-ase). They exist as pro-enzymes (pro-caspases) that need to be activated They include initiator caspases and effector caspases The substrates of the Initiating caspases (Caspases 8, 9 and 10) are effector caspases (caspases 3, 6 and 7). Effectors are responsible for the proteolysis of key cellular proteins, such as actin, nuclear proteins, actins and proteins, nuclear proteins, actin proteins, caspase-activated DNAse (CAD) inhibitor, leaving it free to perform DNA fragmentation (ladder) NUCLEOSOME: The basic unit of the structure of a eukaryotic chromosome. The n. is a globular structure consisting of a cylinder of histones (octamer) wrapped by a stretch of DNA helix.  THE ACTIVATION OF INITIATING CASPASES CAN OCCUR THROUGH TWO MECHANISMS EXTRINSIC PATHWAY (ACTIVATED BY DEATH RECEPTORS) APOPTOSIS THE STIMULUS COMES FROM OUTSIDE MEMBRANE RECEPTORS PLAY AN IMPORTANT ROLE INTRINSECA (MITOCHONDRIAL) PATHWAY of APOPTOSIS DAMAGE ARISES FROM INSIDE THE CELL MITOCHONDRIA PLAY AN IMPORTANT ROLE EXTRINSIC (DEATH RECEPTOR- ACTIVATED) PATHWAY  FASL appartiene alla famiglia del TNF- VIA FAS-FASL: -SI REALIZZA AD OPERA DEI LINFOCITI T CD8 NEI CONFRONTI DI CELLULE INFETTATE DA VIRUS e TUMORALI -COINVOLTA NELL’ELIMINAZIONE DEI LINFOCITI T AUTORESPONSIVI NEL TIMO (SELEZIONE NEGATIVA) INTRINSIC (MITOCHONDRIAL) PATHWAY OF APOPTOSIS Proteins of the Bcl-2 family play an essential role Pro-apoptotic proteins of the Bcl-2 family that have three domains (Bax/Bak) once active form oligomers in the mitochondrial membrane BH3-only proteins can: ACTIVATE BAX/BAK DIRECTLY E.g. Bim, Bid and Puma An active DNA damage p53 that increases the transcription of BAX and BH3-only proteins (e.g. PUMA, NOXA, BID) MOMP: Mitochondrial Outer Membrane Permeability BH3-only proteins can: ACT AS A DEREPRESSOR (frees BH3-only activator proteins from binding to anti-apoptotic proteins) MCP-1 e CX3CL1 1.Initiation: Various intracellular signals trigger the activation of pro-apoptotic proteins (such as Bax and Bak) and the inhibition of anti-apoptotic proteins (such as Bcl-2 and Bcl-xL). This leads to the disruption of the mitochondrial outer membrane. 2.Mitochondrial outer membrane permeabilization (MOMP): The pro-apoptotic proteins Bax and Bak oligomerize and form pores in the outer mitochondrial membrane, leading to the release of pro-apoptotic proteins from the intermembrane space into the cytosol. 3.Release of cytochrome c: Cytochrome c is a key protein released from the mitochondria into the cytosol in response to MOMP. Once in the cytosol, cytochrome c interacts with Apaf-1 (apoptotic protease activating factor 1) and procaspase-9 to form the apoptosome complex. 4.Activation of caspases: The apoptosome complex activates caspase-9, which in turn activates downstream effector caspases such as caspase-3, leading to the execution of apoptosis. Caspases are protease enzymes that cleave various cellular proteins, leading to the characteristic morphological changes associated with apoptosis, such as DNA fragmentation, cell shrinkage, and membrane blebbing. 5.Execution of apoptosis: Once activated, the effector caspases cleave numerous cellular substrates, ultimately leading to the dismantling of the cell and its contents in a controlled and orderly manner. AUTOPHAGY It is estimated that of the 21-22 thousand genes transcribed and translated into proteins, 6500-7000 genes code for proteins that deal with the removal of cellular material. Cell structures are short-lived: proteins can last from 1/2 h to a few days. Protein degradation is essential and involves a number of factors that control gene transcription, regulators of cell proliferation such as the tumor suppressor p53, several proto-oncogenes and antigens in immune system reactions (endogenous antigen presentation in MHC-I) UBIQUITIN-PROTEASOME SYSTEM: degradation and removal of single proteins AUTOPHAGY: concerns protein aggregates and organelles, but also lipids, glycoproteins, mucopolysaccharides UBIQUITIN-PROTEASOME SYSTEM Ubiquitin is a protein found in all eukaryotes Ubiquitin binds to the protein to be degraded in an ATP-dependent pathway using 3 enzymes E1: activates the ubiquitin molecule; this reaction requires energy, released by an ATP molecule E2: binds to activated ubiquitin E3: binds activated ubiquitin to the protein to be degraded AUTOPHAGY = mechanism of cell survival Highly conserved process of degradation of large material in which portions of cytoplasm and organelles (e.g., mitochondria) are sequestered in a double- membrane vesicle (autophagosome) and transported to a degradative organelle (lysosome) for degradation and eventual recycling of the resulting macromolecules. It is a process of self-degradation: the term means "eating oneself" It is a catabolic process for organelles and cytoplasmic material that makes amino acids, nucleotides and fatty acids available for anabolic processes. AUTOPHAGY It depends on the activation of a series of proteins called ATGs (autophagy-related genes), including LC3.  process of autophagy. Autophagosome-lysosome fusion is mediated by a SNARE complex similar to that involved in neuroexocytosis The membrane of the autophagosome is double-membrane: it is believed to result from the winding of cytoplasmic material by a portion of ER that is made in a cistern pattern Eileen White et al. Clin Cancer Res 2015;21:5037-5046 ©2015 by American Association for Cancer Research  Autophagy impairment is also a central mechanism underlying numerous lysosomal storage disorders (LSD) Deficiency of specific lysosomal enzymes in lysosomal storage disorders (LSD) results in the accumulation of undigested material and underlies severe symptoms.  Lysosomal storage diseases Accumulation of undigested material Cell degeneration  Lysosomal Storage Diseases (LSD) Sono definite malattie rare (