Molecular mehanim of cell death By: Sundus Hafeez Rubia ain Momina Masud

CELL DEATH Major cause of cell death: This is due to irreversible cell injury with continuing cell damage, resulting in morphologic changes that can be called as “cell death” Reasons may be Hypoxia Physical agents Chemical agents Infectious agents Genetic imbalances etc

Basically three types 1. Apoptosis= suicide - programmed cell death 2. Necrosis= killing - decay and destruction 3. Autophagy= cell degradation through cell’s own lysosomal machinery

NECROSIS It results in the premature death of cells in living tissue and unregulated digestion of cell components Occurs due to extrinsic factors infection, toxins, or trauma almost always detrimental and can be fatal result in the loss of cell membrane integrity and an uncontrolled release of products of cell death into the intracellular space

Necrosis results in: an inflammatory response in the surrounding tissue Prevention of phagocytes from locating and engulfing dead cells build-up of dead tissue and cell debris at, or near, the site of the cell death

NECROSIS TYPES One of these five macroscopic changes is observed due to necrosis 1. Coagulative necrosis: the outline of the dead cells are maintained and the tissue is firm (due to loss of blood supply) 2. Liquefactive necrosis: the dead cells undergo disintegration and affected tissue is liquefied. 3. Caseous necrosis: combination of coagulative+liquefactive necrosis, resulting in cheese-like mass 4. Fat necrosis: enzymatic digestion of fat by pancreatic enzymes 5. Fibrinoid necrosis: a special form of necrosis usually caused by immune-mediated vascular damage, marked by formation of immune complexes

APOPTOSIS The actual "suicide" of the cell which results in engulfment of the cell remains by specialized immune cells called phagocytes; degradation of engulfed cell. This process helps to eliminate unwanted cells by an internally programmed series of events Balance is maintained between cell proliferation and death

It occurs during: During development for removal of excess cells during embryogenesis To maintain cell population in tissues with high turnover of cells, such as skin, bowels To eliminate immune cells after cytokine depletion, and autoreactive T-cells in developing thymus. Hormone-dependent involution - Endometrium, ovary, breasts, atrophy of ovary during menopause etc. To remove damaged cells by virus To eliminate cells after DNA damage by radiation, cytotoxic agents etc. Cell death in tumors.

APOPTOSIS MORPHOLOGY 1. Shrinkage of cells 2. Condensation of nuclear chormatin peripherally under nuclear membrane 3. Formation of apoptotic bodies by fragmentation of the cells and nuclei. The fragments remain membrane-bound 4. Phagocytosis of apoptotic bodies by adjacent healthy cells or phagocytes 5. Unlike necrosis, apoptosis is not accompanied by inflammatory reaction

Autophagy Autophagy is a self-digesting mechanism responsible for removal of damaged organelles, malformed proteins during biosynthesis, and nonfunctional long-lived proteins by lysosome.

Types Autophagy has been divided into three general types. microautophagy chaperone-mediated autophagy (CMA), macroautophagy.

Microautophagy : cytoplasm material is sequestered through direct invagination to the lysosomal membrane. CMA: proteins flagged with pentapeptide motif (KFERQ) were selectively degraded through direct translocation into lysosome. Macroautophagy : formation of subcellular double-membrane-bound structures called autophagosomes that contain degradable contents of cytoplasm materials and deliver them into lysosomes for breakdown by lysosomal enzymes.

 Autophagy begins with the formation of double-membrane- bounded autophagosomes.  The mammalian target of rapamycin (mTOR) is a negative regulator of autophagosome formation.  Autophagosomes fuse with lysosomes to form autophagolysosomes,  The contents of autophagolysosomes are finally degraded by acidic lysosomal hydrolases.

Type I cell death Apoptosis mechanism

MECHANISM Apoptosis Triggered via Two Pathways Intrinsic or mitochondrial pathway Extrinsic or death receptor pathway

Extrinsic pathway Binding of Fas by FasL induces recruitment of FADD to the cytoplasmic tail of Fas The opposite end of FADD contains a death effector domain (hatched boxes); recruitment of either procaspase-8 or c-FLIP Caspase-8 can cleave Bid truncated Bid (tBid) can inactivate Bcl-2 in the mitochondrial membrane. This allows the escape of cytochrome c, which clusters with Apaf-1 and caspase-9 in the presence of dATP to activate caspase-9. Smac/DIABLO is also released from the mitochondria and inactivates inhibitors of apoptosis (IAPs). breakdown of several cytoskeletal proteins and degradation of the inhibitor of caspase-activated DNase (ICAD).

Intrinsic pathway In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on their surface. Internal damage to the cell (e.g., from reactive oxygen species) causes Bcl-2 to activate a related protein, Bax, which punches holes in the outer mitochondrial membrane, causing cytochrome c to leak out. The released cytochrome c binds to the protein Apaf-1 ("apoptotic protease activating factor-1"). Using the energy provided by ATP, these complexes aggregate to form apoptosomes. The apoptosomes bind to and activate caspase-9. Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7). The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity which leads to digestion of structural proteins in the cytoplasm, degradation of chromosomal DNA, and phagocytosis of the cell.

Necrosis and its Mechanism

Necrosis has been defined as a type of cell death that lacks the features of apoptosis and is usually considered to be uncontrolled. After signaling- or damage-induced lesions, necrosis can include signs of controlled processes such as mitochondrial dysfunction, enhanced generation of reactive oxygen species, ATP depletion and early plasma membrane rupture. The inhibition of specific proteins involved in regulating apoptosis or autophagy can change the type of cell death to necrosis. A classical definition of necrosis based on morphological criteria(early plasma membrane rupture and dilatation of cytoplasmic organelles, in particular mitochondria). necrosis is often associated with unwarranted cell loss in human pathologies and can lead to local inflammation, presumably through the liberation of factors from dead cells that alert the innate immune system

apoptotic cells (which shrink) are engulfed completely by phagocytes, necrotic cells (which swell) are internalized by a macropinocytotic mechanism. meaning that only parts of the cell are taken up by phagocytes necrotic cell death can be a regulated event that contributes to development and to the maintenance of organismal homeostasis. Programmed cell necrosis can be a consequence of extracellular signaling or can be initiated as a form of cellular suicide in response to intracellular perturbations. programmed cell necrosis plays a role in a number of disease processes including vascular-occlusive disease, neurodegenerative diseases, infection, inflammatory diseases, exposures to toxins, and cancer

The core events of necrosis are bioenergetic failure and rapid loss of plasma membrane integrity. These can result from defined molecular events that occur in the dying cell, including increased mitochondrial ROS production, channel- mediated calcium uptake, activation of nonapoptotic proteases, and/or enzymatic destruction of cofactors required for ATP production.

DNA damage-induced necrosis occurs selectively in growing cells. DNA damage-induced necrosis occurs selectively in growing cells. Highly proliferative cells utilize aerobic glycolysis for ATP production as other nutrient sources such as amino acids and lipids are redirected into synthetic reactions that support cell growth and proliferation. In response to alkylating DNA damage, the nuclear enzyme PARP is activated and degrades NAD into poly(ADP)-ribose polymers and nicotinic acid mononucleotide (NAM). The consumption of NAD depletes the cellular NAD pool and shuts down the cell's ability to degrade glucose to support ATP production, leading to necrosis.

Calcium-mediated programmed necrosis. Calcium-mediated programmed necrosis. Intracellular calcium increases in response to the activation of ionotrophic glutamate receptors or through other calcium channels on the plasma membrane or the ER membrane. An intracellular calcium spike induces the activation of Ca2+-dependent proteases and stimulates mitochondrial TCA cycle activity and ROS production. If sustained, the resulting ROS leads to mPT that is dependent on CypD. mPT then leads to the loss of ATP production and necrosis.

Clearance of apoptotic and necrotic cells and its immunological consequences

The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. apoptotic cells engulfment is regulated by a system of receptors on the phagocytic cells. the phagocytic cells detect molecules specific for dying cells. clearance of dying cells is an important fundamental process serving the regulation of normal tissue homeostasis. cell corpses may release cytotoxic substances due to which there are phagocytosed.

binding or uptake of apoptotic cells to phagocytes induces production of transforming growth factor β (TGF- β ) and sometimes interleukin- 10. These anti-inflammatory cytokines have direct autocrine and paracrine effects on proinflammatory cytokine production. Apoptotic cell uptake stimulates lipid mediators such as 15- lipoxygenase and 15-hydroxyeicosatetraenoic acid. This enhances uptake of apoptotic cells by phagocytes. Nonprofessional phagocytes such as endothelial or epithelial cells that phagocytose neighboring apoptotic cells subsequently produce survival and growth factors. These include vascular endothelial growth factor and hepatocyte growth factor. They probably contribute to tissue replenishment and restoration of endothelial and epithelial boundaries.

early apoptotic cells can be cleared silently without release of either pro- or anti-inflammatory mediators. Apoptotic cell uptake predominantly initiates mechanisms that contribute to resolution of injury and repair. but this must be seen in the context of other signals that impinge on the surface receptors of phagocytes. Necrotic cells and pathogens share many of the ligands of apoptotic cells. They usually induce different responses at least partially because they also engage pattern recognition receptors and signaling pathways not activated by apoptotic corpses. apoptotic cell uptake does not immediately switch individual phagocyte function. only does so after a critical number of cells have contributed to an overall change in the microenvironment.