Fribourg 1+2.Pptx

T Kaufmann ‐ Apoptosis (1+2) Nov 16 2009

Apoptosis: Mechanisms, Techniques &Therapeuc Targets

Thomas Kaufmann Instute of Pharmacology University of Bern

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

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STRESS

steady state normal physiological funcon

increased (physiological) adaptaon

excessive cellular damage CELL DEATH

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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

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Cellular stress and damage is reversible up to a certain point: point‐of‐no‐return

Cell Death

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Happy Stressed Cellular Adaptaon cell cell

Cellular Damage

CELL DEATH

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All forms of ssue damage start with damaged single cells

necroc cell

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Two Main Forms of Cell Death

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Outdated Concept…

Apoptosis Necrosis programmed physiological cell death Accidenal acve pathological regulated passive “good” cellular lysis “bad“

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PROGRAMMED CELL DEATH

Apoptosis Pyroptosis

Autophagic Cell Death Pyronecrosis

Necroptosis (Pro‐ Corniﬁcaon grammed Necrosis)

NECROTIC CELL DEATH

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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

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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

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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

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haemorrhagic infarct Severe myocardial ischemic infarct

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• 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

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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

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Stages of Necrosis

F. Chan, Worcester 2009 T Kaufmann ‐ Apoptosis (1+2) 17

Necrosis

S. Marn, Dublin

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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

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Fat ssue necrosis (acute pancreas)

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Coagulave necrosis (skeletal muscle)

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Caseous Necrosis (“Käsige Nekrose”) in lung from tuberculosis paent

‐> “Cheese‐like” appearance of granuloma – typical and only seen in tuberculosis

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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

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Morphological Changes during Apoptosis

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Apoptosis

S Marn, Dublin

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“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

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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

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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

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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

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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

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• 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

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C) Shaping of Immune System

• B Cell Maturaon

Strasser A, Nat Rev Immunol 2005

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• T Cell Maturaon

Strasser A, Nat Rev Immunol 2005

healthy thymus (mouse): ca 20% dead thymocytes

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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)

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Autoimmunity

• Too much apoptosis in healthy cells/ssues

• Not enough apoptosis in immune eﬀector cells

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CTL‐Mediated Tissue Damage

CTL (CD8+ T cell) Target cell

P/G

Perforin/Granzymes

FasL FasL Fas

CTL Target cell

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Perforin/ Granzyme System

Voskoboinik et al. Nat Rev Imm 2006

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Gra‐vs‐Host Disease acute GvHD, skin

Bone Marrow Transplantaon (2008) 41, 1029–1035

CD8+

acute GvHD, colon

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ALPS (autoimmune lymphoproliferave syndrome)

Reduced T and B lymphocyte apoptosis

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Cancer

Hanahan & Weinberg, Cell 2000

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Cancer

• Bcl‐2 staining of paraﬃn embedded human breast carcinoma secon (Epitomics)

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Polyp (Colon Mucosa)

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Necrosis Triggers Inﬂammatory Responses

inﬁltrang neutrophils in necroc ssue (contracon band necrosis)

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Phagocytosis of Apoptoc Cells Prevents Inﬂammaon

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Find‐Me Signals

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Find‐Me Signals

• ATP, UTP <‐> P2Y2 (G‐Protein coupled receptor)

• CX3CL1 <‐> CX3CR1 • Lysophosphadylcholine (LPC) <‐> G2A

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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

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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)

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FLOW CYTOMETRY hp://www.invitrogen.com/site/us/en/home/support/Tutorials.html

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How to Quanfy Apoptosis (I)

PS = phosphadylserine

healthy apoptoc late apoptoc (secondary necrosis)

FITC‐Annexin V Propidium Iodide (PI)

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FITC‐AnnexinV / PI Staining

Jurkat, etoposide 1µg/ml, 24 h

4 Data.009:R0 10

3 10 F L /ml) 3 g 2 - 10 H 1.51 15.8 59.4 23.2 1 10 PI (1.25 µ

0 10 0 1 2 3 4 10 10 10 10 10 FL1-H GFP‐Annexin V

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Caspases are the Central Players of Apoptosis

Initiator Procaspase Initiator Procaspase

Initiator Caspase Initiator Caspase

Effector Procaspases

EFFECTOR CASPASES

Specific proteolytic cleavage of . . vital intracellular targets 2009 T Kaufmann ‐ Apoptosis (1+2) 56

Apoptoc Pathways

Extrinsic

INDIVIDUAL INITIATION Intrinsic

Execuon

COMOOM EXECUTION . .

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Caspases

• Caspase = Cysteine Protease with Asp‐speciﬁcity • Present as inacve Proenzymes • Acvated by proteolyc cleavage • Acvang Caspase‐Cascade • Common execuoner phase

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Proteolyc Processing of Caspases

Thymocytes + apoptoc smulus inacve pro‐form me

prodomain p18 p10 subunit subunit proteolyc processing P10 P20

acve heterotetramer Jost PJ et al., Nature 2009

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A) DEATH RECEPTOR (extrinsic): Fas/CD95, TNF-R1, TRAIL-R1/-R2, ...

DISC DISC: death inducing signalling complex

procaspase-8 (-10)

FLIP caspase-8 (-10)

effector procaspases (-3, -6 + -7)

IAP effector caspases . . (-3, -6, -7)

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A) DEATH RECEPTOR (extrinsic)

DISC B) MITOCHONDRIAL stress signals (stress-induced/ intrinsic) procaspase-8 (-10) Bcl-2 Family

FLIP caspase-8 (-10)

Apoptosome mitochondria effector procaspases Caspase-9 (-3, -6 + -7)

IAP IAP

effector caspases . . (-3, -6, -7)

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The Bcl‐2 Protein Family

Bfk

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Pro‐ An‐ apoptoc apoptoc

Bcl‐2 BH3‐only Mcl‐1

Bax/Bak Bcl‐xL

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The Mitochondrial Apoptotic Pathway is Regulated by Members of the Bcl-2 Protein Family

Bcl-2 Family

Mitochondria

IAP- Cyt.c + antagonists

IAPs

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Cytochrome c Release, PS‐exposure, Secondary Necrosis

Green: cytochrome c

Red: phosphadylserine

Blue: DNA stain

Film SJ Marn, Dublin

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Further Reading

• Kroemer G et al, Cell Death & Differenaon 16 (2009), 3‐11; “Classificaon of cell death: recommendaons of the Nomenclature Commiee on Cell Death 2009” • Raff MC, Nature (1992); 356(6368):397‐400; “Social controls on cell survival and cell death” • Taylor R et al., Nat Rev Mol Cell Biol Vol 9 (2008), p231‐, “Apoptosis, controlled demolion at the cellular level” • Strasser A, Nat Rev Imm (2005) Vol 5, p189‐200 “The Role of BH3‐only Proteins in the Immune System” • Ravichandran KS & Lorenz U, Nat Rev Immun Vol 7 (2007), p 964‐, “Engulfment of apoptoc cells: signals for a good meal” • Oliveira JB et al., J Clin Immunol (2008), 28 Suppl 1: 20‐8, “Disorders of Apoptosis: Mechanisms for Autoimmunity in Primary Immunodeficiency Diseases” • Voskoboinik I et al., Nat Rev Immunol (2006), 6:940‐52, “Perforin‐mediated target‐cell death and immune homeostasis”

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Generation of a Knockout Mouse

1. [Subcloning and] characterisaon of the locus of interest ‐>“in silicio“ (databases. E‐ tools, bioinformacs etc)! 2. Knockout vector construcon (= targeng vector) ‐> from BAC clone (Bacterial Arficial Chromosome) containing whole locus plus large Enough flanking regions (arms) 3. Electroporaon and homologous recombinaon in Embryonic Stem cells (ES cells) ‐> tradionally Sv/129 derived => Sv/129 C57BL/6 mixed genec background ‐> C57BL/6 derived ES cells 4. Southern blots to idenfy correct ES clones (heterozygous!) 5. Blastocyst injecon of ES clones and chimera generaon 6. Breeding to the F1 and F2 generaon ‐> genotyping 7. Breeding to homozygocyty 8. For LoxP‐flanked condional construct: Crossing to Cre+ transgenic strain (e.g. Cre transgene under ssue specific promoter) 2009 T Kaufmann ‐ Apoptosis (1+2) 67

 1 year minimum  expensive (~CHF 30'000) 2009 T Kaufmann ‐ Apoptosis (1+2) 68

Genomic Organisaon of Locus

‐> e.g. ENSEMBL (www.ensembl.org)

‐> genomic organisaon (introns/exons, promoter region, tx start site(s), infos on (alternave?) splicing etc ‐> retrieve sequence (genomic, mRNA/cDNA)

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BACsBACs

BAC: > 10 kb 5‘ and 3‘ of gene of interest AND as small as possible RP23‐262D13 C57BL/6 Balb/c

RP23‐262D13 = ~168 kb ‐> DNA prep (very low copy!), Cam selecon ‐> veriﬁcaon by PCR; e.g. within bok Exon:

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(by homologous recombinaon)

targeted region 5‘ arm 3‘ arm

• 5‘ and 3‘ arm: the longer the beer

• targeted region (to be replaced by neo‐casee): the shorter the beer

• Herpes Simplex Virus Thymidine Kinase: ‐> 'suicide gene'

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Sv/129 or C57BL/6‐derived

+ subcloning

= heterozygous

hp://nobelprize.org/nobel_prizes/medicine/laureates/2007/med07eng.pdf

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HSV‐TK NOT present In recombinant allele! Acve drug Prodrug

ES cell killed

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(Balb/c derived)

C57BL/6 ES cells Balb/c blastocyst ⇒Black & white chimeras

(Chimeras)

Chimera x C57BL/6 wt = heterozygous!

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Conditional Approach using the Cre/LoxP System

‐ LoxP mutant sequences allow single inversion

LoxP sequence: ATAACTTCGTATA ATGTATGC TATACGAAGTTAT

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