1 APOPTOSIS What is it and how does it work at the cellular level? Medi501 - Sept. 20/2012.

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APOPTOSIS

• What is it and how does it work at the cellular level?

Medi501 - Sept. 20/2012

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Objectives of this lecture

• Understanding what apoptosis is – extrinsic vs. intrinsic pathways

• Distinguishing apoptosis from other types of cell death (necrosis, necroptosis, autophagy)

• What key proteins in mitochondria regulate apoptosis

• Functions of BCL-2 family proteins

• Understanding how caspases are regulated

•Assays used to detect/quantify apoptosis

CELL DEATH

Several ways in which cells die:

• Apoptosis - cells are induced to commit suicide; regulated cell death

• Necrosis - death due to injurious agents

• Necroptosis – regulated form of necrosis

• Autophagy leading to cell death

DAMAGED CELLS DIE BY NECROSIS:

- mechanical damage - exposure to toxic chemicals/poisons

A series of changes in cells dying by necrosis:

• they swell (as do organelles like mitochondria) because the ability of the plasma membrane to control the passage of ions and water is disrupted

• cell contents leak out; inflammation of surrounding tissues

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NECROPTOSIS

It became apparent that cells could undergo necrosis in response to cell death receptors

This occurred without activation of caspases, or when caspases were inhibited

In the past few years, it has been discovered that specific kinases (RIPK1, 3) are activated as key markers of cell death by necroptosis

Pathways regulating NECROPTOSIS

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AUTOPHAGY

Autophagy refers to a process whereby cells engulf their own organelles to preserve energy.

Can occur in response to nutrient stress.

Involvement of AMP kinase, which senses elevated levels of AMP - and thus, low ATP

Autophagic cell survival - i.e. as a means of saving the cell until conditions are more favourable.

Autophagic cell death occurs with time - may be hard to distinguish from apoptosis.

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APOPTOSIS

• First described (and named) by Wylie and Kerr in 1973

• An active process directed by inherent mechanisms leading to cell death

• Under normal conditions, cells block apoptosis to survive

• Many ways that apoptosis is initiated

CELLS COMMIT ‘SUICIDE’ - APOPTOSIS:

• cells shrink (ion balance lost)

•Mitochondrial outer membrane breaks down (becomes ‘leaky’); a key event is the release of cytochrome C (it is normally only in mitochondria)

• blebs appear on the cell surface

• chromatin (DNA and protein) is degraded

• cells break into small, membrane-wrapped, fragments

CELLS COMMIT ‘SUICIDE’ - APOPTOSIS:

• Phosphatidylserine (PS), which is in the inner leaflet of the plasma membrane becomes exposed on the surface. • PS serves as one of the ‘signals’ for phagocytic cells like macrophages which then engulf the cell fragments.

• Phagocytic cells secrete cytokines that inhibit inflammation.

• Death by apoptosis is ‘organized’ so it is also called ‘Programmed Cell Death’.

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Signalling pathways regulating apoptosis(Zimmerman et al)

APOPTOSIS - ROLE IN DEVELOPMENT

• resorption of the tadpole tail at the time of its metamorphosis into a frog

• formation of fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them.

• sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation

• formation of the proper connections (synapses) between neurons requires that surplus cells be eliminated (knockout mice lacking some of the key genes for apoptosis die due to large brains)

APOPTOSIS destroys cells that are a threat:

• virus-infected cells killed by cytotoxic T cells

• immune response - effector cells removed to avoid the possibility of attacking self; defects in apoptosis can cause autoimmune diseases

• cells with damaged DNA can disrupt proper embryonic development (birth defects possible); may also become cancerous

• radio- or chemotherapy uses apoptosis to kill cells

Apoptosis in the Regulation of the Hematopoietic system

Daily, our bone marrow produces 1-2 x 1010 granulocytes and approx. 1011 total hemopoietic cells(6.25 Billion cells during this lecture)

Cell death must match cell production.Increases in white cell counts depend on increased levels of survival cytokines.

REGULATION OF APOPTOSISWhat determines when cells will commit to an apoptosis program?

Balance between competing signals:

Positive signals that block apoptosis: Growth (survival) factor Adhesion to substrate Attachment to other cells

These signals transmitted into the cell suppress apoptosis machinery and loss of these positive signals may be enough

REGULATION OF APOPTOSIS

Negative signals that can initiate apoptosis:-internal apoptosis machinery is activated if growth factor signals no longer stop it- molecules that bind to specific receptors (death receptors) on the cell surface and signal the cell to begin the apoptosis program.- increased levels of oxidants within the cell damage the DNA (as can other agents like UV light , X-rays, chemotherapy drugs)

INTERNAL REGULATION OF APOPTOSIS

- BCL-2 family proteins can either be pro-apoptotic or anti-apoptotic

- they regulate mitochondrial (and ER) permeability

-at mitochondria, cytochrome C in the inner mitochondrial space must not leak out to prevent apoptosis

- at the ER, Ca2+ exit is regulated; its presence in cytosol can enhance mitochondrial events

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Signalling pathways regulating apoptosis: Intrinsic pathways (Zimmerman et al)

Members of BCL-2 family

Anti-Apoptosis

Pro-ApoptosisCyto C/

Fragments

SURVIVAL VS. DEATH

THE DUEL BETWEEN SURVIVAL AND DEATH REGULATORS AT THE MITOCHONDRIA

APAF-1

CASPASECASCADE

APOPTOSIS

SURVIVAL

Mitochondria

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CASPASES

Conservedstructural features

Caspase 9 acts in an autocatalytic fashion;Inhibitors of Apoptosis block Capases

by direct Binding; Initiator caspase cleaves and activates effector caspases

Apoptosome

Pro-caspase 9

Pro-caspase 3Pro-caspase 6

etc.

XIAPc-IAP

Survivin

Activecaspase 9

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Signalling pathways regulating apoptosis:Intrinsic pathways (Zimmerman et al)

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DISPLACEMENT MODEL OF BCL-2 FAMILY ACTIONBH3-only proteins displace Bcl-2 pro-survival members

that inhibit Bax and Bak (Leber)

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Direct activation model - BH3 only proteins bind to Bax to promote its activation and membrane insertion

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Embedded Together - Initial activation New model proposed by Leber et al

Bcl-X

Bax

CASPASES

CYTO. C

Regulation of BAD by Phosphorylation

Bax Inhibitorsof Bax

Bax

Activatorsof Bax

BAD

14-3-3

SurvivalKinases

Bcl-X

BADS112

S136

Bax

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Control of Cell survival by PI3K/PKB

Duronio, Biochem J.

EXTERNAL SIGNALS

Fas and the TNF receptor are receptors at the cell surface

- binding of the complementary death activator (FasL and TNF respectively) causes receptor trimerization- this transmits a signal that leads to recruitment and activation of caspase 8 - caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to death of the cell- caspase 8 can also cleave Bid to form tBid which can activate the mitochondrial events

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Signalling pathways regulating apoptosis:Extrinsic pathways (Zimmerman et al)

EXTERNAL SIGNALS

FasL expression is regulated by Forkhead Transcription Factors (e.g. FOXO3a)

FOXO3a is inhibited by phosphorylation mediated by PKB/akt (PI3Kinase-dependent), thus linking pro-survival effects of PI3K/PKB with its inhibitory effects on this death pathway

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Control of Cell survival by PI3K/PKB

Duronio, Biochem J.

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Methods of apoptosis analysis

DNA fragmentation

Flow cytometry to detect cell surface annexin V

Flow cytometry to detect sub-diploid DNA by PI staining (small DNA fragments leak out of cells)

TUNEL staining (detect DNA ends)

Detection of active Caspases by immunoblot or fluorescent substrates

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DNA is cleaved at inter-nucleosomal sites when apoptosis is induced

Increasing conc. of PI3K inhibitor causes apoptosis in cells (left half of gel).

Scheid et al, Biochem. J. 312: 159-62 (1995)

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FLOW CYTOMETER

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Use of Flow cytometry and staining of DNA in permeabilized cells to assay apoptosis

Cytokine starvation induces apoptosis within 24h. Mcl-1 and mutant forms have variable ability to block apoptosis.

Jamil et al, 2010.

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Use of TUNEL (TdT-Mediated dUTP Nick-End Labeling) to detect fragmented DNA in

tissue sections

Normal Apoptosis induced

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Apoptosis analysis

Double staining for annexinV and PI for early and late apoptosis.

Fluorescent substrate analog binds to active caspases.

Green stain indicates active caspases and Hoechst stain shows fragmented nuclei.

Scott et al, J. Orth. Res, 23: 1219-25, 2005.

References

Leber, B, Lin, J and Andrews, DW. Embedded together: The life and death consequences of the Bcl-2 family with membranes. Apoptosis 12, 897-911 (2007).

Danial, NN and Korsmeyer,SJ. Cell death: critical conrol points. Cell 116, 205-219 (2004).

Zimmerman, KC, Bonzon, Green, DR. The machinery of programmed cell death. Pharmacology and Therapeutics 92: 57-70.(2001).

Duronio, V. The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem. J. 415: 333-344 (2008).

Vandenabeele et al, Molecular mechanisms of necroptosis. Nature Rev. Cell Biol. 11, 700-714 (2010).

Flow cytometry tutorial: http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html