Apoptosis, Necroptosis for undergraduate

Apoptosis, Necroptosis for undergraduate

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  APOPTOSIS NECROPTOSIS Dr B Santhoshi 1st YearPathology Resident
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  APOPTOSIS • Apoptosis isa programmed cell death. • It is induced by a tightly regulated suicide program in which the cells that are destined to die activate intrinsic enzymes that degrade the cells genomic DNA and nuclear and cytoplasmic proteins. • Apoptotic cells break up into plasma membrane–bound fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus.
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  • While theplasma membrane remains intact, its surface components are altered so as to produce “find me” and “eat me” signals for phagocytes. • As a result, the dead cell and its fragments are rapidly devoured, before the contents leak out, and therefore apoptosis does not elicit an inflammatory reaction. • Apoptosis was first recognized in 1972 by the distinctive morphologic appearance of membrane-bound fragments derived from cells, and named after the Greek designation for “falling off.”
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  • It wassubsequently discovered in model organisms such as worms that certain cells undergo apoptosis at precise times during development. • This phenomenon, termed programmed cell death, is controlled by the action of a small number of genes and is required for normal embyrogenesis. • Thus, apoptosis is a unique mechanism of cell death, distinct from necrosis in many respects.
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  DIFFERENCES BETWEEN NECROSISAND APOPTOSIS FEATURE NECROSIS APOPTOSIS Cell size Enlarged (Swelling) Reduced (Shrinkage) Nucleus Pyknosis, karyorrhexis, karyolysis Fragmentation into nucleosome size fragments Plasma membrane Disrupted Intact; altered structure Cellular contents Enzymatic digestion; may leak out of cell Intact; may be released into apoptotic bodies Adjacent inflammation Frequent No Physiologic or Pathologic role Usually pathologic (culmination of irreversible injury) Often physiologic (for eliminating unwanted cells) Pathologic after cell injury, DNA damage
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  CAUSES OF APOPTOSIS Apoptosisoccurs in two broad contexts:  As part of normal physiologic processes  As a pathophysiologic mechanism of cell loss in many different diseases.
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  APOPTOSIS IN PHYSIOLOGIC SITUATIONS •Death by apoptosis is a normal phenomenon that serves to eliminate cells that are no longer needed, or as a mechanism to maintain a constant number of various cell populations in tissues. • It is estimated that humans turn over almost 1 million cells per second. • Central to this process is death of cells by apoptosis and their removal by phagocytes.
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  APOPTOSIS IS IMPORTANTIN THE FOLLOWING PHYSIOLOGIC SITUATIONS: • The removal of supernumerary cells (in excess of the required number) during development. • Involution of hormone-dependent tissues on hormone withdrawal. Eg: Endometrial cell breakdown during the menstrual cycle Ovarian follicular atresia in menopause Regression of lactating breast after weaning
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  • Cell turnoverin proliferating cell populations. Eg: Epithelial cells in intestinal crypts to maintain a constant cell number. • Elimination of potentially harmful self-reactive lymphocytes to prevent immune reactions against one’s own tissues. • Death of host cells that have served their useful purpose, such as neutrophils in an acute inflammatory response, and lymphocytes at the end of an immune response.
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  APOPTOSIS IN PATHOLOGIC CONDITIONS •Apoptosis eliminates cells that are injured beyond repair without eliciting a host reaction, thus limiting collateral tissue damage. • Death by apoptosis is responsible for loss of cells in a variety of pathologic states:  DNA Damage by Radiation and cytotoxic anticancer drugs.  Accumulation of misfolded proteins.  Apoptosis can be induced during viral infections.  Apoptosis may also contribute to pathologic atrophy in parenchymal organs after duct obstruction such as in pancreas, parotid gland and
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  MORPHOLOGY Cell shrinkage • Cellsize is reduced, the cytoplasm is dense and eosinophilic and the organelles, although relatively normal, are more tightly packed. • This contrasts with necrosis, in which an early feature is cell swelling, not shrinkage. 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 into two or more fragments.
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  Formation of cytoplasmicblebs and apoptotic bodies • The apoptotic cell first shows extensive surface membrane blebbing. • Followed by fragmentation of the dead cells into membrane bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments. 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.
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  • In H&E-stainedtissue, the apoptotic cell appears as a round or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin. • Because the cell shrinkage and formation of apoptotic bodies are rapid and the pieces are quickly cleared by phagocytes, considerable apoptosis may occur in tissues before it is apparent in histologic sections. • The absence of an inflammatory response can also make it difficult to detect apoptosis by light microscopy.
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  MECHANISMS OF APOPTOSIS •Apoptosis results from the activation of enzymes called caspases (so named because they are proteases containing a cysteine in their active site and cleave proteins after aspartic residues). • Like many proteases, caspases exist as inactive proenzymes and must undergo enzymatic cleavage to become active. • The presence of active caspases is therefore a marker for cells undergoing
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  • The processof apoptosis may be divided into an initiation phase, during which some caspases become catalytically active and unleash a cascade of other caspases. • An execution phase, during which the terminal caspases trigger cellular fragmentation. • Regulation of these enzymes depends on a finely tuned balance between the abundance and activity of pro-apoptotic and anti-apoptotic proteins.
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  • Two distinctpathways converge on caspase activation:  The mitochondrial pathway and  The death receptor pathway. • Although these pathways intersect, they are generally induced under different conditions, involve different initiating molecules, and serve distinct roles in physiology and disease.
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  THE MITOCHONDRIAL (INTRINSIC) PATHWAYOF APOPTOSIS • The mitochondrial pathway is responsible for apoptosis in most physiologic and pathologic situations. • It results from increased permeability of the mitochondrial outer membrane with consequent release of death-inducing (pro-apoptotic) molecules from the mitochondrial intermembrane space into the cytoplasm.
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  • Mitochondria areorganelles that contain remarkable proteins such as cytochrome c, a double edged sword that is essential for producing the energy (e.g., ATP) that sustains cell viability, but when released into the cytoplasm (an indication that the cell is not healthy) initiates the suicide program of apoptosis. • The release of pro-apoptotic proteins such as cytochrome c is determined by the integrity of the outer mitochondrial membrane, which is tightly controlled by the BCL2 family of proteins.
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  • There aremore than 20 members of the BCL family, which can be divided into three groups based on their pro-apoptotic or anti- apoptotic function and the BCL2 homology (BH) domains they possess. Anti-apoptotic: • BCL2, BCL-XL, and MCL1 are the principal members of this group • They possess four BH domains (called BH1-4). • These proteins reside in the outer mitochondrial membrane as well as in the cytosol and ER membranes. • By keeping the mitochondrial outer membrane impermeable, they prevent leakage of cytochrome c and other death-inducing proteins into the cytosol.
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  Pro- apoptotic: • BAXand BAK are the two prototypic members of this group. • They contain the first three BH domains (BH1-3). • On activation, BAX and/or BAK oligomerize within the outer mitochondrial membrane and enhance its permeability. • They form a channel in the outer mitochondrial membrane that allows cytochrome c leakage from the intermembranous space.
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  Regulated apoptosis initiators: •Members of this group, including BAD, BIM, BID, Puma, and Noxa. • Contain only one BH domain, the third of the four BH domains, and hence are sometimes called BH3-only proteins. • The activity of BH3-only proteins is modulated by sensors of cellular stress and damage. • When upregulated and activated, they can initiate apoptosis.
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  • Growth factorsand other survival signals stimulate the production of anti-apoptotic proteins such as BCL2, thus protecting cells from apoptosis. • When cells are deprived of survival signals, suffer DNA damage, or develop ER stress due to the accumulation of misfolded proteins, BH3-only proteins are upregulated through increased transcription and/or post-translational modifications (e.g., phosphorylation). • These BH3-only proteins in turn directly activate the two critical pro-apoptotic family members, BAX and BAK, which form oligomers that insert into the mitochondrial membrane and allow proteins from the inner mitochondrial membrane to leak out into the cytoplasm.
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  • BH3-only proteinsmay also bind to and block the function of BCL2 and BCL-XL. • At the same time, synthesis of BCL2 and BCL-XL may decline because their transcription relies on survival signals. • The net result of BAX-BAK activation coupled with loss of the protective functions of the anti-apoptotic BCL2 family members is the release into the cytoplasm of several mitochondrial proteins such as cytochrome c that can activate the caspase cascade.
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  • Once releasedinto the cytosol, cytochrome c binds to a protein called APAF-1 (apoptosis-activating factor-1), forming a multimeric structure called the apoptosome. • This complex binds to caspase-9, the critical initiator caspase of the mitochondrial pathway, and promotes its autocatalytic cleavage, generating catalytically active forms of the enzyme. • Active caspase-9 then triggers a cascade of caspase activation by cleaving and thereby activating other pro-caspases (such as caspase-3), which mediate the execution phase of apoptosis.
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  • Other mitochondrialproteins like Smac/DIABLO enter the cytoplasm, where they bind to and neutralize cytoplasmic proteins that function as physiologic inhibitors of apoptosis (IAPs). • The normal function of the IAPs is to block the inappropriate activation of caspases, including executioners like caspase-3, and keep cells alive. • Thus, IAP inhibition permits initiation of the caspase cascade.
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  THE EXTRINSIC (DEATHRECEPTOR– INITIATED) PATHWAY OF APOPTOSIS • This pathway is initiated by engagement of plasma membrane death receptors. • Death receptors are members of the tumor necrosis factor (TNF) receptor family that contain a cytoplasmic domain involved in protein- protein interactions. • This death domain is essential for delivering apoptotic signals.
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  • The bestknown death receptors are the type 1 TNF receptor (TNFR1) and a related protein called Fas (CD95). • The mechanism of apoptosis induced by these death receptors is well illustrated by Fas, a death receptor expressed on many cell types. • The ligand for Fas is called Fas ligand (FasL). • FasL is expressed on T cells that recognize self antigens and on some CTLs that kill virus-infected and tumor cells.
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  • When FasLbinds to Fas, three or more molecules of Fas are brought together, and their cytoplasmic death domains form a binding site for an adaptor protein called FADD (Fas-associated death domain). • Once attached to this complex, FADD binds inactivecaspase-8 (or caspase- 10), bringing together multiple caspase molecules and leading to autocatalytic cleavage and generation of active caspase-8. • In turn, active caspase-8 initiates the same executioner caspase sequence as in the mitochondrial pathway.
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  • This extrinsicapoptosis pathway can be inhibited by a protein called FLIP, which binds to procaspase-8, thereby blocking FADD binding, but cannot activate the caspase. • Some viruses and normal cells produce FLIP as a mechanism to protect themselves from Fas mediated apoptosis. • The extrinsic and intrinsic pathways of apoptosis are initiated in fundamentally different ways by distinct molecules, but there may be interconnections between them. • The combined activation of both pathways delivers a fatal blow to the cells.
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  EXECUTION PHASE OFAPOPTOSIS • The intrinsic and extrinsic pathways converge to activate a caspase cascade that mediates the final phase of apoptosis. • The intrinsic mitochondrial pathway activates the initiator caspase-9, whereas the extrinsic death receptor pathway activates caspase-8 and caspase-10.
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  • The activeforms of these caspases trigger the rapid and sequential activation of the executioner caspases, such as caspase-3 and caspase- 6, which then act on many cellular components. • Once activated these caspases cleave an inhibitor of a DNase, making the DNase enzymatically active and allowing DNA degradation to commence. • Caspases also proteolyze structural components of the nuclear matrix and thus promote fragmentation of nuclei.
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  REMOVAL OF DEADCELLS • The formation of apoptotic bodies breaks cells up into “bite-sized” fragments that are edible for phagocytes. • Apoptotic cells and their fragments also undergo several changes in their membranes that actively promote their phagocytosis so they are most often cleared before they lose membrane integrity and release their cellular contents.
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  • In healthycells, phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this phospholipid “flips” out and is expressed on the outer layer of the membrane, where it is recognized by several macrophage receptors. • Apoptotic bodies may also become coated with natural antibodies and proteins of the complement system, notably C1q, which are recognized by phagocytes.
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  • Thus, numerousligands induced on apoptotic cells serve as “eat me” signals and are recognized by receptors on phagocytes that bind and engulf these cells. • This process of apoptotic cell phagocytosis is called efferocytosis; it is so efficient that dead cells disappear, often within minutes, without leaving a trace.
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  • In addition,production of pro-inflammatory cytokines is reduced in macrophages that have ingested apoptotic cells. • Together with rapid clearance, this limits inflammatory reactions, even in the face of extensive apoptosis.
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  NECROPTOSIS • It isa hybrid that shares aspects of both necrosis and apoptosis. • Morphologically, and to some extent biochemically, it resembles necrosis, as both are characterized by loss of ATP, swelling of the cell and organelles, generation of reactive oxygen species (ROS), release of lysosomal enzymes, and ultimately rupture of the plasma membrane.
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  • Mechanistically, itis triggered by signal transduction pathways that culminate in cell death, a feature similar to apoptosis. • Because of these overlapping features, necroptosis is sometimes called programmed necrosis to distinguish it from forms of necrosis driven passively by toxic or ischemic injury to the cell. • In sharp contrast to apoptosis, the signals leading to necroptosis do not result in caspase activation, and hence it is also sometimes referred to as “caspase-independent” programmed cell death.
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  • The processof necroptosis starts in a manner similar to that of the extrinsic form of apoptosis, that is, by ligation of a receptor by its ligand. • Ligation of TNFR1 is the most widely studied model of necroptosis. • Necroptosis involves two kinases called Receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3).
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  • Ligation ofTNFR1 recruits these kinases into a multiprotein complex, and RIPK3 phosphorylates a cytoplasmic protein called MLKL. • In response to its phosphorylation, MLKL monomers assemble into oligomers, translocate from the cytosol to the plasma membrane, and cause the plasma membrane disruption that is characteristic of necrosis. • This explains the morphologic similarity of necroptosis with necrosis
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  • Necroptosis ispostulated to be an important death pathway both in physiologic and pathologic conditions. • For example, physiologic necroptosis occurs during the formation of the mammalian bone growth plate. • In pathologic states, it is associated with cell death in steatohepatitis, acute pancreatitis, ischemia-reperfusion injury, and neurodegenerative diseases such as Parkinson disease. • Necroptosis also acts as a backup mechanism in host defense against certain viruses that encode caspase inhibitors (e.g., cytomegalovirus).
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  PYROPTOSIS • It isa form of apoptosis that is accompanied by the release of the fever-inducing cytokine IL-1 (pyro refers to fever). • Microbial products that enter infected cells are recognized by cytoplasmic innate immune receptors and can activate the multiprotein complex called the inflammasome. • The function of the inflammasome is to activate caspase-1 (also known as interleukin-1β– converting enzyme), which cleaves a precursor form of interleukin-1 (IL-1) and releases its biologically active form.
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  • IL-1 isa mediator of many aspects of inflammation, including leukocyte recruitment and fever. • Caspase-1 and the closely related caspases-4 and -5 also induce death of the cells. • Unlike classical apoptosis, this pathway of cell death is characterized by release of inflammatory mediators. • It is the mechanism by which some microbes cause the death of infected cells and at the same time trigger local inflammation.
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  FERROPTOSIS • It isa distinct form of cell death that is triggered when excessive intracellular levels of iron or reactive oxygen species overwhelm the glutathione-dependent antioxidant defenses to cause unchecked membrane lipid peroxidation. • The widespread peroxidation of lipids disrupts many aspects of membrane function, including fluidity, lipid-protein interactions, ion and nutrient transport, and signaling pathways. • The overall effect is the loss of plasma membrane permeability, which ultimately leads to cell death resembling necrosis.