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Cell Death Dr. Aoife Gowran [email protected] Ext. 1418 Different cells -‐ different life spans WBC 4 days (total) RBC 120 days Neurone 50 -‐ 100 years The Cell Cycle G0 Start point Adapted from Figure 17-‐4 Molecular Biology of the Cell (© Garland Science 2008) Cell cycle – check points Figure 17-‐14 Molecular Biology of the Cell (© Garland Science 2008) Cell cycle control I • Controlled by – Protein kinases • Add a phosphate group onto a protein • Act on and modify the acTvity of proteins • Cyclin dependant protein kinases (Cdk) – Cyclins Figure 17-‐15 Molecular Biology of the Cell (© Garland Science 2008) Cell cycle control III Figure 17-‐16 Molecular Biology of the Cell (© Garland Science 2008) Types of cell death • Programmed cell death – Cells that kill themselves in a controlled way • Apoptosis • Autophagy • Necrosis – Premature death – Cells that die due to injury or trauma Apoptosis Ordered process in response to a Death signal Cell shrinkage + membrane blebbing Membrane integrity is maintained NO rupture of the membrane Organelles targeted nucleus -‐ DNA fragmentaTon mitochondria PhagocyTc clearance Die Developmental apoptosis PROGRAMMED CELL DEATH is essenTal for normal development Digits CNS Figure 18-‐2 Molecular Biology of the Cell (© Garland Science 2008) Immune system Autophagy-‐induced apoptosis Self digesTon (iii) (ii) (i) Parts of the cytoplasm (i), organelles (ii), old proteins (iii) sequestered in autophagic vacuoles Disposal involves the acTon of enzymes in lysosomes May be an a^empt to stay alive during periods of cell starvaTon Ordered process -‐ similariTes with apoptosis i.e. normal degradaTon of cell components no inflammaTon Advantages/Disadvantages Necrosis + -‐ Immediate No going back EffecTve Inflammatory response Apoptosis Autophagy Controlled Inappropriate Precise apoptosis Disposal of waste Can lead to Provides energy cell death Cell death thresholds Necrosis AdapTve change fluctuaTons in pH etc Apoptosis Mild Gross Injury Recovery possible Recovery not possible Adapted from, Raffray and Cohen, 1997 Many Cell Death Types Necrosis Necroptosis Apoptosis** Autophagy-‐induced Apoptosis MitoTc catastrophe Programmed cell death **‘Ectopic expression’ Horwitz, 2007 All remove damaged cells from an organism they just do it in different ways! There is overlap. Apoptosis Summary • • • • • • • • • Cells receive a death signal Undergo morphological changes Shrink and condense Cytoskeleton collapses Nuclear envelope disassembles Nuclear chromaTn condenses and fragments Cell surface ‘blebs’ Breaks up into smaller pieces ‘apoptoTc bodies’ Special cells then engulf them (for example phagocytes) Necrosis Catastrophic failure of homeostasis ATP independent -‐ No protein/mRNA synthesis Swelling of cell and cellular organelles random DNA degradaTon Rupture of cell and release of contents IniTates inflammatory response Necrosis: Tissue consequences The Johns Hopkins University, Division of InfecTous Diseases: h^p://hopkins-‐id.edu/diseases/hepaTTs.html Apoptosis Necrosis Physiological or pathological Always pathological Single cells Sheets of cells Energy dependent Energy independent Cell shrinkage Cell swelling Membrane integrity maintained Membrane integrity lost CharacterisDc nuclear changes Nuclei lost ApoptoDc bodies form Do not form DNA cleavage No DNA cleavage Regulatable process Not regulated EvoluDonarily conserved Not conserved Dead cells ingested by neighbouring cells Dead cells ingested by neutrophils and macrophages What causes a cell to die? OxidaTve stress (ROS) DNA damage Excitotoxins e.g., glutamate, Ca+, Na+ etc. Growth factor withdrawal ChemotherapeuTc drugs Inflammatory mediators e.g., TNF-‐α, IL-‐1β Toxins e.g., β-‐amyloid RadiaTon (uv and γ) Expression of tumour suppressor proteins e.g., p53 ROS – reacDve oxygen species -‐ Derived from endogenous or exogenous sources -‐ e.g., O2.-‐ , .OH and H2O2 -‐ Low levels act as signalling molecules -‐ Higher levels have deleterious effects due to oxidaTve damage to proteins, lipids and DNA -‐ Persistent levels compromises oxidaTve defence mechanisms -‐ Induce apoptosis via mitochondrial uncoupling and death receptor pathways -‐ ROS generaTon mediates cytokine induced apoptosis DNA Damage -‐acTvates either 1) cell cycle arrest and DNA repair or 2) apoptosis if damage is irreparable -‐Death in response to DNA damage iniTated by PI3K e.g., ATM -‐these kinases acTvate check point kinases e.g., ChK1 & ChK2 -‐ ChKs aTvate pro-‐apoptoTc proteins e.g., p53 and caspases PI3K (phosphoinosiTde 3-‐kinase) ATM (ataxia telangiectasia mutated) ChK (checkpoint kinase) Apoptosis Machinery Components Apoptosis triggered by Extrinsic signals Intrinsic signals AcTvaTon of death Release of toxic proteins Receptors -‐ Death Domain from organelles Downstream signalling Cascade of protein-‐protein interacTons Downstream signalling acTvaTon of the Caspase cascade Extrinsic pathway 1. AcDvaDon of death receptors Receptor ligands Transmembrane Belong to TNF family Transmit death signal FasL,TNFα,TRAIL e.g., Fas,TNFR, TrimerisaDon TRAIL-‐R 1&2 2. Recruitment of adapter proteins at death domain 3. Downstream signalling Caspase acTvaTon csp-‐8 ⇒csp-‐3 Extrinsic pathway Common in immune cells CNS -‐ weakly acTve, Fas neurite remodelling Death receptors transmit death signal cell surface ⇒ intracellular p/w Decoy receptors -‐truncaTon of death domain 2 categories of cells Type I/II Depending on downstream signalling mode Type I -‐ efficient caspase acTvaTon Type II -‐ less efficient, needs mitochondrial signalling -‐ “amplifiers” Intrinsic pathway Death signal comes from inside the cell Cell organelles targeted Lysosomes ROS Bax P p53 Apoptosome cathepsins Cyt-c Mitochondria Caspase acTvaTon XX X DNA fragmentation Outer membrane Mitochondria (mt.) Inner membrane Matrix Early inducTon & regulaTon of apoptosis ROS, DNA damage, Fas signalling Release of small pro-‐apoptoTc proteins from mt. inter membrane space. e.g., cytochrome C, Smac/Diablo, AIF etc. Apoptosome formaTon cytochrome C + Apaf-‐1 + csp-‐9 Effector caspases acTvated (csp-‐7/3) PARP, ICAD acTvaTon ⇒ DNA fragmentaTon www.biology4kids.com Mechanisms of release of small pro-‐apoptoDc proteins -‐ inducTon of mt. permeability transiTon (MPT) -‐ involves opening of a mega pore permeability transiTon pore (PTP) -‐ mt. matrix swelling -‐ mt. uncoupling Bcl-‐2 family of proteins: Regulators on the mitochondria Pro-‐ Bax Bak anD-‐ Bcl-‐2 Bcl-‐Xl BH3-‐only Bid Bad Bcl-‐2/ Bax,Bak cycling at the outer mt. membrane Regulated protein−protein interacTons are also key to the understanding of a second set of apoptoTc regulators, the Bcl-‐2 family. This family has been divided into three groups, based on structural similariTes and funcTonal criteria The key funcTon of Bcl-‐2 family members seems to be to regulate the release of pro-‐ apoptoTc factors, in parTcular cytochrome c, from the mitochondrial intermembrane compartment into the cytosol Lysosomes & Other Organelles Early inducTon & regulaTon of apoptosis Targeted rupture Release of small pro-‐apoptoTc proteins e.g., cathepsins Increases cellular pH Effector caspases acTvated (csp-‐3 acTvaTon by cathepsin-‐D) p53 Tumour suppressor p53 p53 p53 P Tetramer phospho-‐protein Regulates gene transcripTon DNA synthesis DNA repair Cell cyle senescence cell death p53 p53 p53 is short lived 1/2 life of 10-‐30 min! Normally present at low levels NegaTve regulated by Mdm2 AcTvated by mulTple mechanisms Associates with organelles Figure 1. Cytosolic and mitochondrial p53 apoptoTc pathways. In the cytosolic p53 apoptoTc pathway, nuclear p53 induces Puma expression, which in turn releases cytosolic p53 held inacTve in the cytoplasm through binding to Bcl-‐XL. Then, cytosolic p53 induces Bax oligomerizaTon and mitochondrial translocaTon. AccumulaTon of p53 in the cytosol as a consequence of normal intracellular transport or stable monoubiquiTnaTon is the major source for mitochondrial p53. In the mitochondria, p53 induces Bax and Bak oligomerizaTon, antagonizes the Bcl-‐2 and Bcl-‐XL anTapoptoTc effect, and forms a complex with cyclophilin D in the mitochondrial inner membrane. These changes result in marked disrupTon of mitochondrial membranes and subsequent release of both soluble and insoluble apoptogenic factors. MPT, mitochondrial permeability transiTon; U, ubiquiTn. (Amaral 2010) Caspases: the central execuDoners (cysteinyl aspartate specific proteases) cysteine proteases -‐ acTve-‐site cysteine, cleave specific substrates at aspartate residues large protein family -‐ 14 to date! AcTvated specifically in apoptoTc cells Caspase-‐mediated cleavage of specific substrates (>100) also explains several of the other characterisTc features of apoptosis Caspase AcTvated DNase (ICAD) cuts genomic DNA between nucleosomes, to generate DNA fragments cleavage Pro acTve InacTve zymogen can be another caspase Act on substrates eg., PARP, Fodrin 2 (main) groups IniTator caspases -‐ acTve at death receptors & mitochondria eg., 8, 10 contain DED domain 2, 9 contain CARD domain Effector caspases -‐ acTve at later stages eg., 3, 6 & 7 DetecTon of cell death Cell morphology Necrosis Apoptosis Reduced cell volume Swollen mitochondria Hoechst Stain • Hoechst 33258 stains nuclei and emits fluorescent signal. • Cells may be viewed under microscope • Normal cells have homogeneous staining • Apoptotic cells have condensed chromatin and DNA fragmentation Plasma membrane NecroTc Dye exclusion tests trypan blue propidium iodide ApoptoTc Annexin V Healthy cell ApoptoTc cell PhosphaTdylserine V V Nucleus/DNA DNA fragmentaTon hallmark of apoptosis Isolate DNA electrophoresis h^p://www.sgul.ac.uk/depts/immunology/%7Edash/apoptosis/ He et al., 2005 Nucleus/DNA DNA fragmentaTon TUNEL stain 3’OH Fragmented DNA Cells in culture Tissue secTons Downer et al., 2007 KulTma et al., 2004 What happens when it all goes wrong? Apoptosis & Disease Humans approximately 1014 cells – each capable of commizng suicide by apoptosis Compromised apoptosis process – inappropriate cell death– disease pathogenesis too li^le / too much PotenTal for therapeuTc use– modulaTng apoptosis for treatment of human diseases Cancer • CumulaTve lifeTme risk – 1 in 3 men – 1 in 4 women • Lung cancer – Breast/prostate – Colorectal Breast cancer cells dividing. David Becker/Wellcome Photo Library Figure 20-‐14 Molecular Biology of the Cell (© Garland Science 2008) High jacked! Cancer • Lifestyle factors (90-‐95%) – Weight/diet/alcohol/ smoking • InfecTous agents – HPV • Environmental factors – RadiaTon/chemical • Inherited mutaTons (5-‐10%) Cancer • Proto oncogene (Gain of funcTon) – normally involved in proliferaTon BUT – Becomes cancer promoTng with mutaTon • Tumour suppressor – normally suppresses tumour BUT – Has a loss of funcTon with mutaTon Cell cycle and Cancer II • Cancer • p53 (nearly all) • Rb (nearly all) • Ras (30%) • Cell cycle checkpoints fail (approx. % in relaTon to adenocarcinoma of the lung) • p53 (50%) • C-‐Myc (10%) • p16 (70%) • Rb (80-‐100%) • Ras (30%) upregulated downregulated Cell cycle and Cancer III • DNA repair enzymes – Excision • Chromosome clock becomes deregulated – hTERT (80%) • Chromosome structural changes – Proteins – TranslocaTons – Loss (3p – 90%) upregulated downregulated Cell death and Cancer I • Programmed cell death – Death receptors (extrinsic pathway) (90%) – p53 (intrinsic pathway) – Caspases (iniTator and execuToner) downregulated Cell death and Cancer II • Mitochondria – Bcl2 (35%) (anT-‐apoptoTc) – Bcl-‐XL (anT-‐apoptoTc) – Bax (pro-‐apoptoTc) • Mitochondrial DNA – Higher mutaTonal rate seen in cancer – Possible link to apoptosis or proliferaTon upregulated downregulated Therapies • Chemotherapy targets: – DNA replicaTon – Spindle formaTon – Cell cycle (anT-‐mitoTc) – Apoptosis • PromoTon of pro-‐apoptoTc proteins • Block of anT-‐apoptoTc proteins • OligonucleoTdes • TargeTng Bcl2 • Gene therapy • TargeTng p53 Apoptosis and neurodegeneraTon Normal Atrophy due to ND www.memorydisorder.org Alzheimer’s Disease (AD) Age-‐associated ND disorder -‐ deficits in memory and intellectual funcToning -‐ demenTa -‐ sporadic and hereditary forms AD features Senile plaques (Aβ) Image credit: Jannis ProducTons. Rebekah Fredenburg, computer animaTon. Pathogenesis of AD -‐ shortage of protecTve soluble amyloid -‐ surplus of neurotoxic Aβ1-‐42 Alzheimer’s Disease Tangles Tau Normal Protein associated with microtubules Phosphorylated by pro-‐apoptoTc proteins e.g., MAP kinases Tau collapses and neurofibrillary tangles form on microtubules Inhibits the movement of essenTal components inside the cell microtubules Image credit: William I. Rosenblum,www.pathology.vcu.edu Alzheimer’s Disease & Apoptosis Suggested by the presence of the following in AD brains Aβ induces ROS, lipid and protein peroxidaTon and decreases the acTvity of anToxidant enzymes Pro-‐apoptoTc proteins Bax c-‐jun N terminal kinase (JNK) Caspase acTvaTon (-‐3, -‐9 and -‐8) DNA fragmentaTon Amyotrophic lateral sclerosis (ALS) Also known as motor neurone disease SelecTve degeneraTon of upper and lower motor neurones Familial cases involve gene mutaTons e.g., in the SOD1 gene (gain of toxic funcTon mutaTon) also mutaTons in mitochondrial DNA Evidence for the involvement of apoptosis Mitochondrial dysfuncTon, oxidaTve stress, DNA fragmentaTon, caspase-‐3, increased Bax and decreased Bcl-‐2 Fas death receptor signalling Parkinson’s disease (PD) Age-‐associated ND disorder -‐ degeneraTon of dopaminergic neurones in substanTa nigra -‐ impaired movement & tremors Pathological hallmark are Lewy bodies -‐ proteinaceous cytoplasmic inclusions made of misfolded proteins e.g., α-‐synuclein Hereditary PD caused by mutaTons in the several genes OxidaTve stress -‐ unambiguously associated with PD oxidaTon of lipids, proteins and DNA ALSO decreases in GSH -‐ vulnerable to toxic insults Role for MAP kinases in PD Reading lists General review arDcles on cell death/apoptosis Lawen A. Apoptosis – an introducTon. BioEssays 2003, 25;888-‐896. Raffray M., and Cohen GM. Apoptosis and Necrosis in Toxicology: A ConTnuum or DisTnct Modes of Cell Death? Pharmocol. Ther., 1997, 75;3:153-‐177. Vandenabeele P., Galluzzi L., Vanden Berghe T., Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat. Rev. Mol. Cell BIol. 2010, 11;10:700-‐14. Reading lists Specific review arDcles Okouchi M., Ekshyyan O., Maracine M., Aw T.Y. Neuronal apoptosis in neurodegeneraTon. An;oxid Redox Signal 2007, 9;1059-‐1096. Vousden K.H., Lane D.P. p53 in health and disease. Nat Rev Mol Cell Biol 2007, 8;275-‐283. Reading lists Website pages For excellent movies illustraTng apoptoTc mechanisms; h^p://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/ InformaTon on cell-‐fate signalling pathways; h^p://www.biocarta.com/genes/Apoptosis.asp 2008 UMass Undergraduate Life Science Research Symposium Keynote Lecture by Dr. Robert Horvitz, 2002 Nobel Laureate in Physiology or Medicine. "Programmed Cell Death in Development and Disease" Skip the first 5 minutes! h^p://video.google.com/videoplay?docid=351110226494524234#
