Apoptosis, Necrosis, and Excitotoxicity - 4BBY1030

L15 4BBY1030: Apoptosis, necrosis and excitotoxicity [email protected] 1 Cell death We recognize different forms of cell death Necrosis Traumatic cell death from acute injury Apoptosis This involves activation of a death programme. Proposed in the 1970s, but general acceptance took another 2...

L15 4BBY1030: Apoptosis, necrosis and excitotoxicity [email protected] 1 Cell death We recognize different forms of cell death Necrosis Traumatic cell death from acute injury Apoptosis This involves activation of a death programme. Proposed in the 1970s, but general acceptance took another 20 years. A specialized form of cell death that is confined to one type of tissue: Excitotoxicity In neural tissue only 4 Reluctance to admit such a thing was possible since cells go to such great lengths to maintain their own viability that it was difficult to believe that a mechanism would evolve that enables a cell to commit suicide. Everyday we turnover billions of cells. quick, efficient, and immunologically silent Comparison of necrosis with apoptosis I. causes Apoptosis Necrosis Withdrawal of growth Injury/insult factors ischemia Chemotherapy hypoxia Contact with cytotoxic T cells Following a developmental programme Ischemia – a restriction of blood supply to tissues depriving tem of nutrients (principally glucose) and oxygen Hypoxia – deprivation of oxygen only Cytotoxic T cells which are are part of the immune system and kill cancer cells and infected cells (principally cells infected with viruses). The cancer cells and infected cells present antigens on their surfaces, these are Recognised by cytotoxic T cells, the contact between the cytotoxic T cells and the target cells activates the apoptotic pathway within the target cells. Not the only mechanism used by Cytostolic T cells, they can release a protein called perforin which forms pores on the membranes of target cells, which disturbs cellular homeostasis and kills them (which would be a type of necrosis) Comparison of necrosis with apoptosis II. Characteristics Apoptosis Necrosis Membrane damage Intact membrane (with blebbing) Blebbing: when the cytoskeleton separates from the cell membrane, causing the membrane to form spherical protrusions (blebs) The McGraw-Hill Companies, Inc. http://www.cyto.purdue.edu/cdroms/cyto4/15_apop/data/malorni/malorni.htm Comparison of necrosis with apoptosis II. Characteristics Apoptosis Necrosis Membrane damage Intact membrane (with blebbing) Chromatin flocculation Chromatin condensation Comparison of necrosis with apoptosis II. Characteristics Apoptosis Necrosis Energy levels rapidly depleted Leakage of cellular contents Energy levels maintained (or depleted slowly) No leakage Elicits an inflammatory No inflammatory response response Apoptotic cells rapidly engulfed by phagocytes (before they lyse and spill contents) Apoptosis follows a pre-determined path apoptotic bodies phagocytic cell cell fragmentation chromatin condensation membrane blebbing http://cc.scu.edu.cn/G2S phagocytosis Cell has undergone PCD, and has then been engulfed by a phagocytic cell* *Preventing release of intracellular molecules is particularly important in the nervous system, where release of excitotoxic mediators (e.g. glutamate) from dying cells can cause injury to adjacent neurons. Cell has undergone PCD, and has then been engulfed by a phagocytic cell* Necrotic cell seems to have exploded *Preventing release of intracellular molecules is particularly important in the nervous system, where release of excitotoxic mediators (e.g. glutamate) from dying cells can cause injury to adjacent neurons. Why cells commit apoptosis ① During metamorphosis Vogt, 1842: noted that resorption of the notochord and its replacement by vertebrae, involved physiological cell death (tadpole gills lost also). Lokshin and Williams, 1964: described regulated cell death during insect metamorphosis Noticed by Vogt in 1842…noted cell death happens in a predictable pattern during amphibian metmaorphosis with the Process beginning with the disappearance of tadpole tails. And Lokshin and Wiliams were the first to use the phrase programmed cell death when describing insect metamorphosis. Ordinary tadpoles have gills. During the tadpole stage they 'breathe' like a fish, by passing oxygenated water through their gills and absorbing the oxygen. Frogs,, have lungs and breathe like we do Why cells commit apoptosis ② Elimination of cells that have served their purpose during development e.g. cells that have sculpted hands and feet: Mouse paw with interdigital tissue (webbed). This tissue does not appear in the adult mouse One day later Why cells commit apoptosis ③ Cells infected by viruses ④ Cancer cells ⑤ Cells bearing excessive DNA damage ⑥ To promote self tolerance. Autoreactive lymphocytes undergo apoptosis before they fully develop. The common theme is removal of unwanted cells The biochemical characteristics of apoptosis 1. DNA is cleaved by an endonuclease to give a ladder pattern when the DNA is resolved by electrophoresis. DNA isolated from mouse thymus lymphocytes, after induction of apoptosis. Fragments are distinct in size because cleavage occurs in linker regions between nucleosomes. Figure 18-4a Molecular Biology of the Cell (© Garland Science 2008) Cells treated with an antibody that activates a death receptor The biochemical characteristics of apoptosis Fragmentation means that many new free ends of DNA will be generated. These can be detected by the TUNEL assay Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labelling The transferase recognizes the free ends and adds dUTPs that are labelled with a marker. Figure 18-4b Molecular Biology of the Cell (© Garland Science 2008) The biochemical characteristics of apoptosis 2. phosphatidylserine is exclusively located on the inner leaflet of the plasma membrane lipid bilayer In apoptotic cells, phosphatidyleserine flips to the outer leaflet. …where it can be detected by labelled annexin V http://www.biocat.com/cgi-bin/page/sub2.pl? main_group=cell_biology&sub1=apoptosis&sub2=apoptosis_detection_%28phosphatidylserin/ annexin_based%29 Externalized phosphatidylserine is an ‘eat me’ signal apoptotic cells release ‘find me’ signals, attracting motile phagocytes Receptors on the phagocytes bind to externalized phosphatidylserine, stimulating: o release of antinflammatory cytokines o engulfment of the dying cell Engulfed corpse processed Ruchandran (2010) J Exp Med v207 p1807 Find me signals low levels of nucleotides ATP and UTP,, lysophosphatidylcholine, or sphingosine 1-phosphate Engagement of the engulfment receptors stimulates release of antinflammatory cytokines such as TGF-b, IL-10, and prostaglandin E2 (PGE2). where the engulfed corpse goes through a series of phagosome maturation steps, eventually leading to its degradation The biochemical characteristics of apoptosis 3. Apoptotic cells lose the electrochemical potential that exists across the inner mitochondrial membrane. The change in membrane potential can be measured using positively charged fluorescent dyes. Evolutionary conservation of the apoptotic pathway Caenorhabditis elegans (hermaphrodite) has 959 somatic cells, but during development 131 undergo apoptosis. Four genes were identified that controlled this. This was the first evidence that apoptosis was under the control of a genetic programme. The same genes, encoding Caspases (a class of protease) are found in humans where they play a similar role. 2 During development 1090 somatic cells are generated for each hermaphrodite, of which 131 invariantly undergo apoptosis Caspases are the enzymes that drive apoptosis in multicellular eukaryotes. Proteases with Cysteine at their active site, which cleave their substrates at specific aspartate sites There are >10 Caspase genes in the human genome Examples of Caspase targets: ICAD, inhibitor of Caspase dependent DNAase. Digestion of ICAD renders the DNAase active CAD CAD ICAD 3 Could mention nuclear localization once ICAD digested ICAD binds to the catalytic site of CAD Lamins, scaffold proteins of the nuclear envelope (leads to nuclear shrinkage and fragmentation) Responsible, in part, for the apoptotic DNA cleavage. Figure 18-4a Molecular Biology of the Cell (© Garland Science 2008) 4 Slide 22 Examples of Caspase targets: Structural proteins e.g. Lamins (scaffold proteins of nuclear envelope). Cleavage of lamins leads to nuclear shrinkage and fragmentation Gelsolin (a regulator of actin filament assembly/disassembly). Cleavage of gelsolin leads to membrane blebbing Caspases cause rapid cell death: ▪ They are found in all mammalian cells. ▪ Their premature activation would be lethal. ▪ Robust mechanisms are in place to control activation, including: o synthesis of caspases as inactive zymogens. o highly evolved upstream regulatory pathways including the presence of endogenous inhibitors. There are two apoptotic pathways: I. extrinsic pathway: Responding to extracellular signals, indicating that a specific cell is no longer needed for the well-being of the organism. Involves transmembrane death receptors which are members of the tumor necrosis factor (TNF) receptor superfamily. Sometimes called the death receptor pathway. Members of this receptor family bind to extrinsic ligands and transduce intracellular signals that activate caspases. These cells are no longer needed II. Intrinsic pathway: Apoptotic stimuli cause mitochondrial membranes to become leaky, leading to release of cytochrome c into the cytoplasm. Cytochrome c activates a caspase. Sometimes called the mitochondrial pathway. Responsive to Cytotoxic drugs that have entered the cell DNA damage If the damage can’t be repaired, it is best to dispose of the cell (it could become a tumour cell and threaten the whole organism). ‘Sunburn’ is apoptosis owing to excessive DNA damage (by UV irradiation). Damage caused by UV irradiation UV-C (180-290 nm) - not found in daylight as it is absorbed by the ozone layer - the most energetic and lethal (used as a sterilization agent) UV-B (290-320 nm)- major mutagenic fraction of sunlight induces chemical bonds between adjacent thymines (to give thymine dimers), distorting DNA and causing problems during DNA replication, often resulting in point mutations. UVB causes sunburns and UVA makes you tan. When special receptors in your skin detect UVA radiation, they produce extra melanin. DNA damage leads to UVB-induced apoptosis No insult HeLa cells Piva et al., (2012) Int. J. Mol. Sci 13, 2650-2675 Chromatin condensation Apoptotic bodies DNA damage leads to UVB-induced apoptosis No insult HeLa cells UVB irradiation A pathway that limits development of skin cancer (especially melanoma). Piva et al., (2012) Int. J. Mol. Sci 13, 2650-2675 Chromatin condensation Apoptotic bodies Both extrinsic and intrinsic pathways involve caspases Apoptosis Apoptosis is subject to extensive research because either excessive or insufficient apoptosis can contribute to disease. e.g. of excessive Heart attacks and strokes can feature loss of cells by apoptosis (blocking this could save the cells). Type I diabetes mellitus: underlying cause is apoptosis of pancreatic β-cells (ability to produce insulin now lost). Apoptosis is subject to extensive research because either excessive or insufficient apoptosis can contribute to disease. e.g. of insufficient Autoimmune diseases are characterized by large numbers of lymphocytes in spleen and lymph glands. Stimulating the loss of these cells by apoptosis could limit the extent of the reaction against the individuals own tissues. Apoptosis is usually activated in tumour cells. Defective apoptosis means that cancer will develop and progress. Excitotoxicity Glutamate is the most abundant neurotransmitter in the brain; but it plays pivotal role in the pathogenesis of neuronal cell death. In 1969 Olney coined the term excitotoxicity, to describe when excessive glutamate acts on an excitatory receptors and causes cell death. This is due to an increase in intracellular Ca2+ To understand this, we first review steps involved in synthesis and release of glutamate: Glutamate is synthesized in the brain in two ways. First, from a precursor in the Krebs cycle. The glutamate is then taken up by exocytic vesicles. The second process involves what happens to glutamate after it has been used as a neurotransmitter: http://what-when-how.com/neuroscience/neurotransmitters-the-neuron-part-2/ Glutamate is abundant in nervous system and especially prominent in the brain It is the body’s most prominent neurotransmitter Glia non-neuronal cells that maintain homeostasis, form myelin and provide support and protection for neurons in the central nervous system And peripheral nervous system Myelin is a lipid rich (fatty) substance that surrounds nerve cells and axons, to insulate them ① & ② Nerve terminals and glial cells reuptake released glutamate, via membrane transporters ③ In the glia, glutamate is converted to glutamine. ④ Glutamine is transported from glia into the neuronal terminal via transporters in glial and neuronal terminal membranes 6 ⑤ In the neuronal terminal, glutamine converted to glutamate. ⑥ Glutamate is taken up into vesicles and stored, then released by exocytosis. Released glutamate is actively taken up by the glia and neuronal terminals via glutamate transporters. In the neuronal terminal it is re-packaged into vesicles for subsequent use, in the glia itis converted to glutamine (as in step1). Excitotoxicity Normally, glutamate concentration inside the cells is 10,000x greater than outside. This isn’t a problem as it is sequestered within vesicles. This can be disturbed e.g. during hypoxia, hypoglycaemia, when an excess of glutamate is released. Leading to a prolonged activation of receptors, causing cell death (hence ‘excitotoxicity’) *only applies to cells with glutamate receptors on their surfaces. Why is prolonged activation of receptors dangerous? ① Glutamate binds to NMDA (N-methyl-D-aspartate) and AMPA (α-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid) receptors. ② Activated AMPA receptors allow Na+ to enter the cell, depolarizing the plasma membrane. ③ This dislodges Mg2+ from Biological Psychology, Breedlove and the NMDA receptor, Watson publ. Sinauer Associates (2013) permitting entry of Ca2+ Prolonged exposure to glutamate leads to prolonged entry of Ca2+ NMDA and AMPA receptors, named after artificial agonists that activate them Both are glutamate receptors NMDA receptors are double gated, open when two conditions are simutaneously satisfied, glutamate must be bound to the receptor and the membrane must be depolarized. The second condition is requred for releasing Mg2+ that normally blocks the NMDA chanell So NMDA receptors are only activated when AMPA receptors are activated as well and depolarize the membrane Leads to activation of of Ca2+ dependent enzymes involved in breakdown of Protein Phospholipids Nucleic acid Plus activation of enzymes that lead to elevated levels of reactive oxygen species (ROS), which also react with the above biomolecules. Excitotoxicity could be the final destructive pathway associated with a number of disorders including: stroke, trauma, epilepsy, & neurodegenerative disorders Huntington’s disease. Parkinson’s disease. Alzheimer’s disease. Summary There are different forms of cell death, including necrosis and apoptosis Necrosis is cell death arising from acute cell injury Apoptosis is highly regulated and controlled and leads to death of unwanted cells Excitotoxicity is a type of cell death confined to neural tissue. Elevated levels of the neurotransmitter glutamate causes prolonged activation of glutamatedependent receptors, leading to elevated levels of intracellular Ca2+.