52 - Apoptosis (CSL)

Embed Size (px)

Text of 52 - Apoptosis (CSL)

  • 7/27/2019 52 - Apoptosis (CSL)

    1/48

    Apoptosis

    C.S. Little, PhD

    January 4, 2011

    Lecture 52

  • 7/27/2019 52 - Apoptosis (CSL)

    2/48

    C.S. Little, PhD

    Office: 303 Evans Hall

    Phone: 215-871-6882

    Email: [email protected]

  • 7/27/2019 52 - Apoptosis (CSL)

    3/48

    Reading

    Robbins: Pathologic Basis of Disease. 8th

    Edition. Chapter 1, pp. 11-32.

  • 7/27/2019 52 - Apoptosis (CSL)

    4/48

    Apoptosis

    First described in 1972

    From the Greek, meaning falling off

    cell death that:

    Occurs normally Eliminates unwanted or potentially harmful cells

    Cells that have outlived their usefulness

    Pathologic event

    Cells damaged beyond repair (especially to DNA)

  • 7/27/2019 52 - Apoptosis (CSL)

    5/48

    Apoptosis in Physiologic

    circumstances Embryogenesis

    Implantation

    Organogenesis

    Developmental involution

    metamorphosis

    hormone dependent involution in the adult

    regression of lactating breast after weaning

    endometrial cell breakdown during menstrual cycle ovarian follicular atresia in the menopause

  • 7/27/2019 52 - Apoptosis (CSL)

    6/48

    Apoptosis in physiologic

    circumstances Cell deletion in proliferating cell populations

    intestinal crypt epithelia

    Maintenance of constant cell number

    Death of host cells that have served their purpose

    Neutrophil death in acute inflammation

    Lymphocyte death at the end of an immune response

    Occurs as a result of deprivation of necessary survivalsignals

  • 7/27/2019 52 - Apoptosis (CSL)

    7/48

    Apoptosis in physiologic

    circumstances

    Elimination of potentially harmful self reactive

    (autoimmune) lymphocytes

    During or after maturation

    Cell death induced by cytotoxic Tlymphocytes

  • 7/27/2019 52 - Apoptosis (CSL)

    8/48

    Apoptosis in pathologic

    circumstances

    Cell death induced by a variety of injurious stimuli

    Radiation

    Anti-neoplastic drugs

    Both cause DNA damage

    If repair mechanisms fail, cell undergoes apoptosis

    Heat (mild)

    Hypoxia (mild)

    Stress to the endoplasmic reticulum Accumulation of unfolded proteins

  • 7/27/2019 52 - Apoptosis (CSL)

    9/48

    Apoptosis in pathologic

    circumstances Cell injury in certain viral diseases

    Viral hepatitis

    Pathologic changes in parenchymal organs

    after duct obstruction Pancreas

    Kidney

    Parotid gland

    Cell death in tumors

  • 7/27/2019 52 - Apoptosis (CSL)

    10/48

    The Study of Apoptosis

    131 of 1090 somatic cells generated during development of

    this organism are eliminated by apoptosis.Caenorhabditis elegansCED-3: caspase homologueCED-4: Apaf-1 homologueCED-9: bcl-2 homologue

  • 7/27/2019 52 - Apoptosis (CSL)

    11/48

    Morphology of apoptotic cells:

    Cell shrinkage

    dense cytoplasm

    relative maintenance of cell organelles

    But more tightly packed

    Chromatin condensation Highly characteristic of apoptosis

    chromatin aggregates peripherally around the nuclearmembrane

    eventually nucleus may break up into two or morefragments

  • 7/27/2019 52 - Apoptosis (CSL)

    12/48

    Morphology of apoptotic cells

    Formation ofcytoplasmic blebs and apoptotic

    bodies

    apoptotic bodies composed of cytoplasm and

    tightly packed organelles sometimes nuclear fragments

    Phagocytosis of apoptotic cells or bodies

    NO INFLAMMATION

  • 7/27/2019 52 - Apoptosis (CSL)

    13/48

    EM of apoptotic cell

  • 7/27/2019 52 - Apoptosis (CSL)

    14/48

    EM of late apoptotic cell

  • 7/27/2019 52 - Apoptosis (CSL)

    15/48

    EM of necrotic cell

    Note

    Chromatin clumping

    Organelle swelling

  • 7/27/2019 52 - Apoptosis (CSL)

    16/48

    Biochemical features of apoptosis

    Protein cleavage

    Through activation ofcaspases

    Targets include

    Lamins

    Nuclear scaffold

    Cytoskeleton

    DNAses

    DNA breakdown

    Phagocytic recognition

  • 7/27/2019 52 - Apoptosis (CSL)

    17/48

    Mediators of apoptosis

    CASPASES

    What are caspases?

    Cysteine proteases that cleave after aspartic

    acid residue (Asp-X)At least 10 members have been identified

    Highly conserved across species

  • 7/27/2019 52 - Apoptosis (CSL)

    18/48

    Mediators of apoptosis

    CASPASES

    Involvement at two or more levels:

    initiator caspases: involved in

    decision/commitment to apoptosis (alreadydiscussed)

    Caspase 8, 10 and 9

    effector or executioner caspases: involvedin execution of apoptosis

    Caspase 3, 6 and 7

  • 7/27/2019 52 - Apoptosis (CSL)

    19/48

    Mediators of apoptosis

    CASPASES

    Exist in pro-enzyme or zymogen form

    Undergo cleavage to be activated

    By other caspases or autocatalytically

  • 7/27/2019 52 - Apoptosis (CSL)

    20/48

    Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM)

    2004 Elsevier

    Figure 1-24 p. 28

  • 7/27/2019 52 - Apoptosis (CSL)

    21/48

    The extrinsic apoptotic pathway

    death receptor: ligand interaction

    TNFR superfamily

    Contain cytoplasmic death domains that

    delivers the apoptotic signal

    TNF-R

    Fas

  • 7/27/2019 52 - Apoptosis (CSL)

    22/48

    The extrinsic apoptotic pathway :Fas

    Three or more Fasmolecules become cross-linked by the Fas ligand

    Fas cytoplasmic death

    domains form a binding sitefor an adaptor protein(FADD)

    Also contains a deathdomain

    See Figure 1-26 p. 29

  • 7/27/2019 52 - Apoptosis (CSL)

    23/48

    The extrinsic apoptotic pathway :Fas

    Complex of death receptorsand FADD binds inactivecaspase-8 (pro-caspase-8)via death domains

    Multiple pro-caspase-8

    molecules are brought intoproximity and cleave oneanother to generate activecaspase-8

    Figure 1-29 p.30

  • 7/27/2019 52 - Apoptosis (CSL)

    24/48

    The extrinsic apoptotic pathway :Fas

    Caspase 8 then triggers acascade of caspase activation

    These other caspases mediatethe execution phase ofapoptosis

    This pathway can be inhibitedby FLIP

    Binds to pro-caspase 8 butcannot cleave and activate itbecause it has no enzymaticactivity

    Produced by viruses andsome normal cells

  • 7/27/2019 52 - Apoptosis (CSL)

    25/48

    Extrinsic apoptotic pathway: TNF-R

    death receptor: ligand interaction (TNF: TNF-R)

    TNF is an important mediator of inflammatory

    processes

    Also induces apoptosis

    When TNF binds its receptor, cascade is very similar:

    Leads to association of the receptor with the adaptor

    protein TRADD

    TRADD binds to FADD

    Caspase activation

  • 7/27/2019 52 - Apoptosis (CSL)

    26/48

    Signalling pathways that initiate

    apoptosis: TNF-R

    Major functions of TNF are not by induction of

    apoptosis

    But instead by activation of a potent

    transcriptional factor called NF-kB NF-kB and its inhibitor (IkB)

    Important in regulating cell survival and

    inflammation

  • 7/27/2019 52 - Apoptosis (CSL)

    27/48

    Signalling pathways that initiate

    apoptosis

    So how can TNF induce apoptosis AND

    promote cell survival?

    Which adaptor proteins attach to the TNF-R

    TRADD and FADD: apoptosis TRAFs (TNF receptor associated factors) favor

    activation of NF-kB

  • 7/27/2019 52 - Apoptosis (CSL)

    28/48

    Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 2 December 2004 07:30 PM)

    2004 Elsevier

    Figure 1-30 p.30 (Robbins 7th Edition)

    Intrinsic pathway of apoptosis

  • 7/27/2019 52 - Apoptosis (CSL)

    29/48

    Intrinsic or mitochondrial pathway

    The bcl-2 family

    There are about 20 proteins in this family

    All regulate apoptosis

    Main ones that are anti-apoptotic

    Bcl-2 Bcl-x

    Normally reside in mitochondrial membranes andin the cytosol

    When cells are deprived of survival signals or are

    subjected to stress, these proteins are lost fromthe mitochondrial membrane and are replaced bythe pro-apoptotic members of the family (Baxand Bak)

  • 7/27/2019 52 - Apoptosis (CSL)

    30/48

    Intrinsic or mitochondrial pathway

    pro-apoptotic members of the Bcl-2 family

    Include Bax and Bak

    When Bcl-2/Bcl-x levels decrease,permeability of the mitochondrial membrane

    increases

    Causes leakage of proteins that can activatecaspases

    Best known: cytochrome c

    Important for role in mitochondrial respiration

  • 7/27/2019 52 - Apoptosis (CSL)

    31/48

    Intrinsic or mitochondrial pathway

    Result of increasedmitochondrialpermeability

    And release ofpro-

    apoptotic molecules intothe cytosol

    No known role for deathreceptors

  • 7/27/2019 52 - Apoptosis (CSL)

    32/48

    Intrinsic or mitochondrial pathway

    cytochrome c (continued)

    Once out in the cytosol, cytochrome c binds toapaf-1 (apoptosis-protease activating factor 1;ced-4 homologue)

    Complex of apaf-1 and cytochrome c activatescaspase 9

    Bcl-2 and Bcl-x may also directly inhibit apaf-1activation

    Other mitochondrial proteins such as apoptosisinducing factor (AIF) can enter the cytosol

    Bind to and neutralize inhibitors of apoptosis

    Net effect: initiation of the caspase cascade

  • 7/27/2019 52 - Apoptosis (CSL)

    33/48

    Intrinsic or mitochondrial pathway

    Growth factors andother signals stimulate

    the production ofanti-apoptotic member ofthe Bcl-2 family ofproteins

  • 7/27/2019 52 - Apoptosis (CSL)

    34/48

    Intrinsic or mitochondrial pathway

    The essence of the intrinsic

    pathway is the balance of

    pro- and anti-apoptotic

    molecules that

    Regulate mitochondrial

    permeability

    And release of death

    inducers that are

    normally sequestered inthe mitochondria

  • 7/27/2019 52 - Apoptosis (CSL)

    35/48

    Intrinsic or mitochondrial pathway

    There is evidence that the intrinsic pathway can betriggered without a role for the mitochondria

    Not well-defined

    May also be overlap between the extrinsic and intrinsic

    pathways (may not be distinct) Example: in hepatocytes Fas signalling activates pro-

    apoptotic bid which activates the mitochondrialpathway

  • 7/27/2019 52 - Apoptosis (CSL)

    36/48

    Execution of apoptosis: Role of

    caspases

    Nuclear targets

    Proteins involved in

    transcription (c-Myc and NF-kB-inhibited)

    DNA replication DNA repair poly ADP-ribose polymerase(PARP)

    disassembly of cell structure

    nuclear lamina (keratins 18, 19 and vimentin)

    elements of the cellular cytoskeleton (b-catenin-

    disrupting cell-cell interactions, fodrin and

    gelsolin-disrupting the actin filament network)

  • 7/27/2019 52 - Apoptosis (CSL)

    37/48

    Execution of apoptosis: Role of

    caspases

    activate endonuclease

    Caspase 3

    Activates DNA fragmentation factor 45 (DFF45)

    Activation of DFF45 in turn activates DFF40which plays a critical role in the internucleosomal

    DNA degradation

    Also acts on mitochondrial substrates

    disrupts electron transport

    loss of mitochondrial transmembrane potential

  • 7/27/2019 52 - Apoptosis (CSL)

    38/48

    Execution of apoptosis: Role of

    the endonuclease DNA breakdown

    Cleaves in internucleosomal

    spacer regions

    first into large chunks (50-

    300kb) then into multiples of 180 bp

    Form a ladder on agarose

    gels

  • 7/27/2019 52 - Apoptosis (CSL)

    39/48

    Final stage: phagocytosis

    Apoptotic cells and their fragments have

    markers on their surface that facilitate early

    recognition by nearby phagocytic cells

    The loss of phospholipid asymmetry in theplasma membrane and translocation of

    phosphotidyl serine (PtdSer) to the outer

    leaflet of the lipid bilayer

    very efficient no inflammation

  • 7/27/2019 52 - Apoptosis (CSL)

    40/48

    Phagocytosis of apoptotic cells

    PtdSer and annexin I co-localize on the

    surface of the outer membrane

    Serve as eat me trigger

    phagocyte recognition uptake

    Other cell surface/surface associated

    molecules enhance uptake including

    Mannose binding lectins (MBL) and C1q

  • 7/27/2019 52 - Apoptosis (CSL)

    41/48

    Limited inflammation

    Uptake of apoptotic cells has been shown to

    result in the release of

    IL-10, TGFb, and PGE2

    These are anti-inflammatory mediators

  • 7/27/2019 52 - Apoptosis (CSL)

    42/48

    DNA-damaged mediated apoptosis

    Involves p53

    accumulates when DNA is damaged (such asfollowing ionizing radiation)

    arrests the cell cycle at the G1/S boundary toallow for repair

    if repair fails, p53 triggers apoptosis

    if p53 absent or mutated, favors survival

    p53 may up-regulate Bax, Fas and APAF-1 Activate caspases and cause apoptosis

  • 7/27/2019 52 - Apoptosis (CSL)

    43/48

    CTL-mediated lysis

    Cytotoxic T cells recognize foreign antigens

    presented by Class I MHC at the cell surface

    Upon recognition, CTL secrete

    Perforin: transmembrane pore-formingmolecule

    Granzyme B: serine protease

    Entry facilitated by perforin

  • 7/27/2019 52 - Apoptosis (CSL)

    44/48

    CTL-mediated lysis

    Granzyme B: serine protease

    Cleaves proteins at aspartate residues

    Activates caspases

    By-passes up-stream signalling eventsActs directly by inducing execution phase

    CTL also express Fas-L on their surface andcan induce cell death via this pathway

  • 7/27/2019 52 - Apoptosis (CSL)

    45/48

    Disordered apoptosis and disease

    Disorders associated with inhibited apoptosis

    and increased cell survival

    accumulated cells can give rise to cancer

    p53 mutation hormone-dependent (breast, prostate, ovary)

    autoimmunity

    Individuals with complement component C1q

    defects have an increased risk of developingsystemic lupus erythematosus (SLE) and

    glomerulonephritis

  • 7/27/2019 52 - Apoptosis (CSL)

    46/48

    Disordered apoptosis and disease

    Disorders associated with increased

    apoptosis and excessive cell death

    neurodegenerative diseases

    spinal muscular atrophies ischemic injury

    myocardial infarction

    virus-induced lymphocyte depletion

    AIDS

  • 7/27/2019 52 - Apoptosis (CSL)

    47/48

    Apoptosis and Alzheimers disease research

    From: BMC Neurosci. 2008 Jan 24;9:13.

  • 7/27/2019 52 - Apoptosis (CSL)

    48/48

    Apoptosis and Alzheimers disease research