T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Apoptosis: Mechanisms, Techniques &Therapeuc Targets Thomas Kaufmann Instute of Pharmacology University of Bern [email protected] www.pki.unibe.ch Contents 1 & 2 3 & 4 3 & 4 7 & 8 • Necrosis • Caspases • Bcl‐2 Family • Apoptosis in • Apoptosis • Acvaon of Proteins Disease • Main • Detecon • Death • Therapeuc Apoptoc Receptors Targets Pathways 2009 T Kaufmann ‐ Apoptosis (1+2) 2 STRESS steady state normal physiological funcon increased (physiological) adaptaon excessive cellular damage CELL DEATH 2009 T Kaufmann ‐ Apoptosis (1+2) 3 1 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Cellular Adaptaon to Stress Atrophy • Reducon in cell size and funcon Hypertrophy • Increase in cell size and funcon Hyperplasia • Increase in cell number • Replacement by a more stress resistant cell Metaplasia type • Change of size, form and organisaon Dysplasia • Start of neoplasc transformaons • Genec component; clonal 2009 T Kaufmann ‐ Apoptosis (1+2) 4 Cellular stress and damage is reversible up to a certain point: point‐of‐no‐return Cell Death 2009 T Kaufmann ‐ Apoptosis (1+2) 5 Happy Stressed Cellular Adaptaon cell cell Cellular Damage CELL DEATH 2009 T Kaufmann ‐ Apoptosis (1+2) 6 2 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 All forms of ssue damage start with damaged single cells necroc cell 2009 T Kaufmann ‐ Apoptosis (1+2) 7 Two Main Forms of Cell Death 2009 T Kaufmann ‐ Apoptosis (1+2) 8 Outdated Concept… Apoptosis Necrosis programmed physiological cell death Accidenal acve pathological regulated passive “good” cellular lysis “bad“ 2009 T Kaufmann ‐ Apoptosis (1+2) 9 3 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 PROGRAMMED CELL DEATH Apoptosis Pyroptosis Autophagic Cell Death Pyronecrosis Necroptosis (Pro‐ Cornificaon grammed Necrosis) NECROTIC CELL DEATH 2009 T Kaufmann ‐ Apoptosis (1+2) 10 When is a Cell Dead? When the researcher says so (?) In vitro In vivo • Loss of plasma membrane • Cell (or its fragments) has integrity been engulfed by adjacent and/or cells • Complete fragmentaon of cell and/or nucleus in discrete bodies and/or • Degradaon/fragmentaon of genomic DNA 2009 T Kaufmann ‐ Apoptosis (1+2) 11 Necrosis • Classical Necrosis is always pathological • Passive form of cell death resulng from irreversible cellular damage • Energy independent • Lysis of the cell membrane (and organelles) • Oen whole parts of ssue/organ affected • Triggers inflammatory reacons 2009 T Kaufmann ‐ Apoptosis (1+2) 12 4 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Classical Necroc Stresses • Lack of energy(/blood/oxygen) supply (ischemia) ‐> lack of energy producon • Chemical Stress: azide, inhibitors of respiratory chain many chemodrugs (high doses) oxygen radicals ‐> membrane damage, protein oxidaon • Physical Stress: strong radiaon (UV, γ), heat, cold 2009 T Kaufmann ‐ Apoptosis (1+2) 13 haemorrhagic infarct Severe myocardial ischemic infarct 2009 T Kaufmann ‐ Apoptosis (1+2) 14 • The nature and/or the intensity of the stress determines how a cell dies Stress X Stress Y Healthy Apoptosis Necrosis e.g. γ‐radiaon 2009 T Kaufmann ‐ Apoptosis (1+2) 15 5 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Necrosis Physical damage Chemical ATP depleon damage Cellular homeostasis↓ Membrane potenal↓ Membrane/ energy↓ organelle damage Ion channels↓ Calcium↑ Lysis of cell H20↑ Cellular volume↑ Inflammaon 2009 T Kaufmann ‐ Apoptosis (1+2) 16 Stages of Necrosis F. Chan, Worcester 2009 T Kaufmann ‐ Apoptosis (1+2) 17 Necrosis S. Marn, Dublin 2009 T Kaufmann ‐ Apoptosis (1+2) 18 6 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Morphological Disncons of Necrosis Fat Tissue „Stress“ Necrosis Acvaon of lipases Irreversible Cellular Damage Microbial Denaturing of proteins degradaon (heat/cold/acids) Enzymac degradaon Coagulave Necrosis Caseous Necrosis Colliquave Necrosis „Mummificaon“ Gangrene „Liquefacon“ Putrefacon 2009 T Kaufmann ‐ Apoptosis (1+2) 19 Fat ssue necrosis (acute pancreas) 2009 T Kaufmann ‐ Apoptosis (1+2) 20 Coagulave necrosis (skeletal muscle) 2009 T Kaufmann ‐ Apoptosis (1+2) 21 7 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Caseous Necrosis (“Käsige Nekrose”) in lung from tuberculosis paent ‐> “Cheese‐like” appearance of granuloma – typical and only seen in tuberculosis 2009 T Kaufmann ‐ Apoptosis (1+2) 22 Apoptosis • Physiological AND pathophysiological form of cell death • Programmed cell death • Evoluonary conservaon • Highly regulated and energy‐consuming process • Cells remain intact no inflammatory • Rapid phagocytosis (in vivo) response • Inducon of Tolerance • Fragmentaon of genomic DNA • Oen single cell death 2009 T Kaufmann ‐ Apoptosis (1+2) 23 Morphological Changes during Apoptosis 2009 T Kaufmann ‐ Apoptosis (1+2) 24 8 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Apoptosis S Marn, Dublin 2009 T Kaufmann ‐ Apoptosis (1+2) 25 “Death by Default” (M. Raff, 1992) • Every nucleated cell of our body is programmed to die <=> cell has to be kept alive constantly Pro‐ Pro‐ apoptoc survival Sm X Stress A cell Sm Y Cell Stress B Sm Z 2009 T Kaufmann ‐ Apoptosis (1+2) 26 Inducon of Apoptosis • DNA‐damaging radiaon (UVR, • Growth factor (cytokine) IR) withdrawal • Most chemodrugs • ‘death‐by‐neglect’ (thymocytes) • Viral Infecons (hepas, HIV) • Cell detachment induced death • Inflammaon, sepsis (Anoikis) • Transplant rejecon • Loss of nerve cell connecons • Negave selecon (thymocytes) • Steroids • Oxygen stress 2009 T Kaufmann ‐ Apoptosis (1+2) 27 9 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Apoptotic Cell Death is Essential in a Healthy Organism • Development (embryogenesis) • Tissue homeostasis of adult organism • Shaping of immune repertoire • Terminaon of immune response ‐> apoptosis guarantees the safe re‐ moval of old, unwanted or poten‐ ally dangerous cells 2009 T Kaufmann ‐ Apoptosis (1+2) 28 A) Apoptosis in Development Removal of interdigital webs Syndactyly Hutcheson et al. JEM 2005 2009 T Kaufmann ‐ Apoptosis (1+2) 29 Development of the brain ca. half of the neurons (~ 1012 !) inially produced in the nervous system will be removed by apoptosis during development wildtype apaf‐1‐/‐ Yoshida et al, Cell 1998 2009 T Kaufmann ‐ Apoptosis (1+2) 30 10 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 B) Tissue Homeostasis ~ 1 x 1014 cells ~ 200 cell types > 1 x 106 cells turned over per second! • Colon epithelium – Shedded off every 3 days 2009 T Kaufmann ‐ Apoptosis (1+2) 31 • Haematopoiec system ‐> ca. 0.85 x 109 neutrophilic granulocytes (neutrophils) produced every day per kg body weight! Male of 80 kg: 8x1010/day ca. 8x105/second! Half‐life in blood ca 12 hours 2009 T Kaufmann ‐ Apoptosis (1+2) 32 C) Shaping of Immune System • B Cell Maturaon Strasser A, Nat Rev Immunol 2005 2009 T Kaufmann ‐ Apoptosis (1+2) 33 11 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 • T Cell Maturaon Strasser A, Nat Rev Immunol 2005 healthy thymus (mouse): ca 20% dead thymocytes 2009 T Kaufmann ‐ Apoptosis (1+2) 34 D) Terminaon of Immune Response Effector cell apoptosis Effector cell expansion Viral killing Viral replicaon CD8 T‐cell numbers Viral clearance CTL response Viral tre Infecon Time post infecon Pellegrini et al., TCB 2004 2009 T Kaufmann ‐ Apoptosis (1+2) 35 Aberrant Apoptosis is a Cause or Consequence of Many Diseases • too much apoptosis • too lile apoptosis – Auommune disorders (e.g. pancreac β‐cells) – CANCER – HIV (CD4+) – Hepas (viral, alcohol – Autoimmune disorders induced, …) – Sepsis (immuno‐ suppressed state) – Viral diseases (e.g. EBV) – Neurodegeneraon – Radiaon damage (skin, bone marrow, stem cells) 2009 T Kaufmann ‐ Apoptosis (1+2) 36 12 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Autoimmunity • Too much apoptosis in healthy cells/ssues • Not enough apoptosis in immune effector cells 2009 T Kaufmann ‐ Apoptosis (1+2) 37 CTL‐Mediated Tissue Damage CTL (CD8+ T cell) Target cell P/G Perforin/Granzymes FasL FasL Fas CTL Target cell 2009 T Kaufmann ‐ Apoptosis (1+2) 38 Perforin/ Granzyme System Voskoboinik et al. Nat Rev Imm 2006 2009 T Kaufmann ‐ Apoptosis (1+2) 39 13 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Gra‐vs‐Host Disease acute GvHD, skin Bone Marrow Transplantaon (2008) 41, 1029–1035 CD8+ acute GvHD, colon 2009 T Kaufmann ‐ Apoptosis (1+2) 40 ALPS (autoimmune lymphoproliferave syndrome) Reduced T and B lymphocyte apoptosis 2009 T Kaufmann ‐ Apoptosis (1+2) 41 Cancer Hanahan & Weinberg, Cell 2000 2009 T Kaufmann ‐ Apoptosis (1+2) 42 14 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Cancer • Bcl‐2 staining of paraffin embedded human breast carcinoma secon (Epitomics) 2009 T Kaufmann ‐ Apoptosis (1+2) 43 Polyp (Colon Mucosa) 2009 T Kaufmann ‐ Apoptosis (1+2) 44 Necrosis Triggers Inflammatory Responses infiltrang neutrophils in necroc ssue (contracon band necrosis) 2009 T Kaufmann ‐ Apoptosis (1+2) 45 15 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Phagocytosis of Apoptoc Cells Prevents Inflammaon 2009 T Kaufmann ‐ Apoptosis (1+2) 46 Find‐Me Signals 2009 T Kaufmann ‐ Apoptosis (1+2) 47 Find‐Me Signals • ATP, UTP <‐> P2Y2 (G‐Protein coupled receptor) • CX3CL1 <‐> CX3CR1 • Lysophosphadylcholine (LPC) <‐> G2A 2009 T Kaufmann ‐ Apoptosis (1+2) 48 16 T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009 Eat‐Me Signals • Apoptoc cells display “eat‐me signals” on their surface ‐> phagocytosis by professional phagocytes (mainly macrophages) Ravichandran et al, Nat Rev Immunol 2007 2009 T Kaufmann ‐ Apoptosis (1+2) 49 Macrophage with engulfed apoptoc cells (red) 2009 T Kaufmann ‐ Apoptosis (1+2) 50 Redistribuon of Phosphadylserine in the Plasma Membrane Lipid Bilayer AnnexinV outer leaflet inner leaflet healthy apoptoc PS (phosphadylserine) 2009 T Kaufmann ‐ Apoptosis (1+2) 51 17 T Kaufmann