The Cell - Week 5 PDF

Apoptosis and Necrosis Apoptosis- programmed cell death Necrosis – necrotic cell death in response to cell injury caused by external factors such as toxins trauma and infection – unregulated from cell death which releases all complex machinery in the cell into surrounding tissues. Injury it se...

Apoptosis and Necrosis Apoptosis- programmed cell death Necrosis – necrotic cell death in response to cell injury caused by external factors such as toxins trauma and infection – unregulated from cell death which releases all complex machinery in the cell into surrounding tissues. Injury it self is only occurrent when cell under stressphysical/chemical. Severe – irreversible – cell death Cellular injury Oxygen deprivation: Hypoxia is a deficiency of oxygen that can result in a reduction in aerobic oxidative respiration. Extremely important common cause of cell injury/cell death. Causes include reduced blood flow (ischemia), inadequate oxygenation of the blood, decreased blood oxygencarrying capacity. Reduced oxidative phosphorylation - With resultant depletion of energy stores in the form of adenosine triphosphate (ATP) Cellular swelling caused by changes in ion concentrations and water influx Physical agents: - Mechanical trauma Extremes of temperature (burns and deep cold) Sudden changes in atmospheric pressure Radiation, and electric shock. Chemical Agents and Drugs Infectious Agents Immunologic Reactions Genetic Derangements Nutritional Imbalances Protein-calorie and/or vitamin deficiencies. Nutritional excesses (overnutrition) Necrotic cell death : 1. Swelling of the endoplasmic reticulum and membrane blebing in response to minor trauma 2. If trauma persists, plasma membranes breakdown and the cell contents leaks out. 3. Organelles breakdown, also leaking out their contents 4. In response pro-inflammatory macrophages are recruited, eliciting an inflammatory response. 5. Release of “volatile” intracellular components can lead to significant damage to the surrounding cell population, exacerbating the effect. 6. These Chemotactic factors lead to neutrophil infiltration to degrade dead cells Apoptotic cell death: 1. Chromatin condenses, ready for packaging and degradation 2. DNA cleaved into discreet 200 base pair units. 3. Membrane blebs 4. Intracellular components, including organelles, are packaged into miniature membrane pockets called apoptotic bodies. 5. This process is tightly regulated through a number of cell signalling mechanisms 6. Apoptotic bodies are then engulfed by phagocytes, which degrade the contents and recycle what they can. NECROISIS 6 TYPES OF NECROISIS… Coagulative necrosis. Coagulative necrosis is a type of accidental cell death typically caused by ischemia or infarction. This type of necrosis tends to occur in tissues such as Heart, kidney and adrenal glands Liquefactive necrosis. Liquefactive necrosis involves the transformation of cellular mass into a liquid viscous mass. Commonly associated with bacterial and fungal infections Caseous necrosis. Caseous necrosis is a form of cell death in which the tissue maintains a cheese-like appearance. The dead tissue appears as a soft and white proteinaceous dead cell mass. Fat necrosis. Fat necrosis is a condition that occurs when a person experiences an injury to an area of fatty tissue. This can result in the fat being replaced with the oily contents of fat cells. Fibroid necrosis. Fibrinoid necrosis is a form of necrosis characterised by accumulation of amorphous, basic, proteinaceous material in the tissue matrix Gangrenous necrosis. Gangrenous necrosis refers to the death of tissue due to a lack of blood flow Morphology of necrosis : The morphologic appearance of necrosis is the result of denaturation of intracellular proteins and enzymatic digestion. Necrotic cells are unable to maintain membrane integrity and their contents often leak out, a process that may elicit inflammation in the surrounding tissue. The enzymes that digest the necrotic cell are derived from the lysosomes of the dying cells themselves and from the lysosomes of leukocytes that are called in as part of the inflammatory reaction. Digestion of cellular contents and the host response may take hours to develop. The earliest histologic evidence of necrosis may not become apparent until 4 to 12 hours. Necrosis of Cytoplasm: Increased eosinophilia in H+E stains – attributes in part of the loss of cytoplasmic RNA – part of denatured cytoplasmic proteins When enzymes have digested the cytoplasmic organelles, the cytoplasm becomes vacuolated and appears moth-eaten. Dead cells may be replaced by large, phospholipid masses called myelin figures that are derived from damaged cell membranes. These phospholipid precipitates are then either phagocytosed by other cells or further degraded into fatty acids; calcification of such fatty acid residues results in the generation of calcium soaps. Thus, the dead cells may ultimately become calcified. Necrosis Nuclear material: Nuclear changes appear in one of three patterns Karyolysis: the chromatin fades, which appears to reflect loss of DNA because of enzymatic degradation by due to endonucleases. Pyknosis: characterized by nuclear shrinkage.  Karyorrhexis: the pyknoticnucleus undergoes fragmentation. With the passage of time (a day or two), the nucleus in the necrotic cell totally disappears. Coagulative necrosis – Ischemia Mitochondrial damage leads to significant decrease in oxidative phosphorylation Reduction in ATP production enhances anaerobic glycolysis, as well as impacting on sodium ion pumps. Accumulation of calcium and sodium ions, combined with water retention leads to cellular swelling Accumulation of lactic acid and a decrease (more acidic) cellular pH leads to chromatin clumping Detachment of ribosomes form the ER reduces protein synthesis  Activation of hydrolytic enzymes results in breakdown of the cellular machinery in an uncontrolled manner. Phospholipases breakdown membranes Proteases breakdown and disrupt cytoskeletal proteins Endonucleases breakdown genomic and mitochondrial DNA Mitochondria compromised and leads to the release of pro-apoptotic signals The eventual outcome is necrosis Apoptosis – Programmed cell death Pathway of cell death induced by a tightly regulated suicide program. Controlled by specific genes. Fragmentation of DNA. Fragmentation of nucleus. Blebs form and apoptotic bodies are released. Apoptotic bodies are phagocytized. The rate of apoptosis differs depending on the tissue the cells reside in Apoptosis is a basic requirement during the development of an embryo. Initial development of appendages requires the removal of webbing in between them. This process is tightly regulated through apoptotic signalling In adult humans, apoptosis is occurring continuously. The rate of apoptosis is usually the same as the rate of cell renewal and growth. Apoptosis itself activates in response to either: Withdrawal of survival factors Stimulation by death factors Key definitions: - Caspase – family of protein that important for programmed cell death as well as having potential role in inflammatory responses. 2 main categories of Caspases: Procaspase – Non-active caspases containing inhibitory domains Cytochrome C – Key protein in progression of intrinsic apoptotic pathway Initiator caspases Caspase 2 – Functionally poorly defined, however believed to have role in apoptosis induction following metabolic imbalances Caspase 8 – Key caspase in transmission of signal from extracellular signals. Caspase 9 – Cleaves Caspase 3, 6 and 7 initiating the caspase cascade Caspase 10 – Cleaves and activates caspases 3, 6, 7, 8 and 9. Executioner caspases Caspase 3 - Involved in the activation cascade of caspases responsible for apoptosis execution. Cleaves and activates caspase-6, -7 and -9 Caspase 6 - Involved in the activation cascade of caspases responsible for apoptosis execution. Cleaves poly(ADP-ribose) polymerase in vitro, as well as lamins. Caspase 7 - Cleaves and activates sterol regulatory element binding proteins (SREBPs). Proteolytically cleaves poly(ADP-ribose) polymerase (PARP) Extrinsic – death receptor mediated pathway : - Involves the use of transmembrane receptor mediated interactions. - Collectively called “death receptors”, these receptors are part of the tumour necrosis family of proteins (TNF). They contain 2 distinctive domains A cysteine rich extracellular domain An 80 amino acid cytoplasmic “Death domain” - The cysteine rich extracellular domain allows for the binding of extracellular “death signals”, with the intracellular domain being responsible for transmission of the “death signal” through to the cytoplasm. - Following receptor binding, cytoplasmic adapter proteins are recruited which provide death domains that bind with intracellular death receptors. - For Fas receptors: - Fas receptor binding recruits FADD (Fas associated death domain) - For TNF receptors - TNF ligand binding results in the binding of TRADD (TNF-receptor associated death domain) and the recruitment of FADD and RIP (Receptor-interacting serine/threonine-protein kinase) Intrinsic – Mitochondria Mediated pathway: The intrinsic apoptotic pathway is mediated by the mitochondria. Mitochondria produce both pro and anti-apoptotic factors depending on the scenario they are faced with All proteins within the intrinsic pathway, both pro and anti-apoptotic, are part of the Bcl-2 family of proteins. The key proteins are: Bcl-2 Bax Bad Bcl-x Bak Within the intrinsic pathway, pro survival factors are absent. The lack of pro survival signals leads to the production of death promoting proteins. Death promoting proteins (Bax, Bak) are held in equilibrium with antiapoptotic factors (Bcl-2, Bcl-x). If the balance shifts towards proapoptotic proteins, then the cell is more likely to undergo apoptotic cell death. If apoptosis is favoured, mitochondria release cytochrome C into the cytosol Under normal conditions, cytochrome C is bound to the inner mitochondrial membrane via cardiolipin. In Apoptotic scenarios, mitochondrial ROS stimulates cardiolipin oxidation via cardiolipin specific oxygenase. This releases cytochrome C from its anchor, and allows for progression of the apoptotic signal  Following mitochondrial release of cytochrome C, the next stage of the process is the formation of an Apoptosome. The Apoptosome requires the addition of cytochrome C to an adaptor protein, Apaf-1. This final process is regulated by IAPs (Inhibitor of apoptosis proteins). If the inhibitory signal is not sufficient, effector caspases are activated. Cytochrome C binding promotes the initial formation, and with the addition of procaspase 9 (and activation to form caspase 9) allows progression of the apoptosis signal towards the effector caspases. Execution phase – Irrespective of which pathway which is used to reach this point, the two pathways converge into the execution phase.  Within the extrinsic pathway, executioner caspases are activated by caspase 8 and caspase 10 Within the intrinsic pathway caspase 9 is required through the formation of the Apoptosome. Irrespective of which pathway which is used to reach this point, the two pathways converge into the execution phase. Within the extrinsic pathway, executioner caspases are activated by caspase 8 and caspase 10 Within the intrinsic pathway caspase 9 is required through the formation of the Apoptosome. The caspase that is considered most important in the execution phase is caspase 3. Caspase 3 cleaves ICAD (Inhibited Caspase activated DNase), producing CAD, which is responsible for the fragmentation of the DNA in a very controlled manner. The protein cleaves DNA but also results in chromatin condensation Cytoskeletal reorganisation and disintegration is mediated by a number of proteins, one of which is Gelsolin. Gelsolin is cleaved by caspase 3, which in turn allows Gelsolin to cleave actin filaments. The process requires Ca2+ presence. One of the hallmarks of caspase mediated DNA degradation is the formation of DNA ladder of fragments differing in length by ∼200 base pairs.  This produces a unique laddering effect when run on an electrophoresis gel. Once all of the DNA has been degraded, and the proteins broken down, the next step is the formation of apoptotic bodies and subsequent destruction by phagocytosis. This begins with the blebbing of the membrane.