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

2. 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

3. Several ways in which cells die:
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
Necrosis is the death of cells due to external forces; Apoptosis is cell death that is programmed from within the genome, carried out by intrinsic enzymes; Necroptosis is initiated at the mitochondria, but utilizes alternate mechanisms; Autophagy is engulfement of organelles to destroy them, which can recycle cell components to save the cell in times of starvation, but eventually can lead to cell death

4. 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

5. 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

6. Pathways regulating NECROPTOSIS

7. 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.
Autophagy is an important process, particular under conditions of metabolic stress. It is considered an ancient process that all eukaryotic cells may use.

8. 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

9. 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

10. 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’.

11. Signalling pathways regulating apoptosis
(Zimmerman et al)

12. 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)
Highlighting a few examples of how apoptosis is important in development. There are many cells made at various stages of animal development that are no longer needed and these must be removed in a controlled manner.

13. 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

14. Apoptosis in the Regulation of the Hematopoietic system
Daily, our bone marrow produces 1-2 x 1010 granulocytes and approx 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.
The dynamic changes in the production and removal of cells of the blood must be carefully balanced. While this may seem wasteful, the ability to rapidly produce many inflammatory cells is important for immune responses - when these cells are needed due to activated cells producing key cytokines, the cytokines provide the signals to block apoptosis and thus allow the cells to accumulate at sites of infection (ie. one sees an elevation in the number of white blood cells).

15. REGULATION OF APOPTOSIS
What 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

16. 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)

17. 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
For most discussions of apoptosis, the regulation of mitochondrial integrity is highlighted. It is largely controlled by the activity of BCL-2 family proteins, which are regulated by many processes. We must also remember that much of the BCL-2 family proteins’ activity occurs at the endoplasmic reticulum, also regulating permeability of those membranes.

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

19. Members of BCL-2 family
The pro-survival members of the BCL-2 family act to maintain mitochondrial integrity, largely by blocking the inherent activity of the pro-apoptotic BAX sub- family to cause leakiness of the mitochondria. All of these have multiple BH domains that fold to form structures that regulate the binding interactions among the proteins. In the BH3 sub-family, the BH3 domain also forms a structure that fits into the BH pockets of the other proteins. Different members of this latter group can either act to block the activity of the pro-survival proteins, or they can promote the pro-apoptotic activity of the BAX sub-family.

20. THE DUEL BETWEEN SURVIVAL AND DEATH REGULATORS
SURVIVAL VS. DEATH
THE DUEL BETWEEN SURVIVAL AND DEATH REGULATORS
AT THE MITOCHONDRIA
SURVIVAL
Pro-Apoptosis
Cyto C/
Fragments
Anti-Apoptosis
APAF-1
A simplified schematic that summarizes how the BCL-2 family proteins are working.
CASPASE
CASCADE
Mitochondria
APOPTOSIS

21. CASPASES
Conserved
structural features

22. Caspase 9 acts in an autocatalytic fashion;
Inhibitors of Apoptosis block Capases
by direct Binding; Initiator caspase cleaves and activates effector caspases
Pro-
caspase 9
Active
caspase 9
XIAP
c-IAP
Survivin
Pro-caspase 3
Pro-caspase 6
etc.
The family of proteins known as ‘Inhibitors of Apoptosis’, or IAP’s have the ability to bind to caspases and block their activity. This is thought to be important to prevent inappropriate activation of caspases. If over-produced, the IAP’s may serve to block apoptosis and thus can be a factor in a cell becoming oncogenic. The initiator caspases (mainly 8 and 9) cleave themselves to become active. Then, they can cleave other pro-caspase members to release their inhibitory domains and activate them.
Apoptosome

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

24. DISPLACEMENT MODEL OF BCL-2 FAMILY ACTION
BH3-only proteins displace Bcl-2 pro-survival members that inhibit Bax and Bak (Leber)
This and the subsequent figures are from the review by Leber and Andrews. It provides a comprehensive description of how BCL-2 family proteins may be regulated at a membrane surface. This cartoon shows how small BH3-only proteins compete for binding of the pro-survival proteins (on the left) and thus leave BAX molecules to aggregate together to form pores.

25. Direct activation model - BH3 only proteins bind to Bax to promote its activation and membrane insertion
This model explains how some of the BH3-only proteins seem to be able to directly activate BAX and promote its aggregation. This happens when other BH3 proteins bind to Bcl-XL to inhibit its ability to block the Bax activators.

26. Embedded Together - Initial activation
New model proposed by Leber et al
This additional model is proposed bases on biophysical studies showing that tBID can displace a BAX inhibitor in the cytoplasm and then become embedded in the membrane together with BAX.

27. Regulation of BAD by Phosphorylation
Bax
Inhibitors
of Bax
Activators
of Bax
S112
CASPASES
Survival
Kinases
14-3-3
BAD
S136
CYTO. C
Bcl-X
BAD
Bcl-X
Bax
Bax
Bax
BAD is a highly regulated protein, which is phosphorylated at a number of different sites by several different kinases. Those kinase activities are dependent upon growth factor or cytokine signals. When the BAD becomes dephosphorylated because the kinases are no longer active, then it can bind to BCL-XL. If BAD is bound to BCL- XL, the BCL-XL can no longer bind to BAX, thus allowing BAX to oligomerize and cause pore formation in the membrane.

28. Control of Cell survival by PI3K/PKB
The PI3K signalling pathway regulates multiple downstream events that directly or indirectly have an effect on regulating cell survival.It is now well accepted that the PI3K/PKB pathway plays a key role in cellular survival as well as cell proliferation and thus any improper regulation has important implications in cancer.
Duronio,
Biochem J. 36

29. 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

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

31. 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

32. Control of Cell survival by PI3K/PKB
Duronio,
Biochem J. 36

33. 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
Apoptosis is measured by a number of methods - the most useful of these are quantitative measures that can be used to detect the extent of apoptosis among multiple samples. For analysis of populations of cells, the flow cytometer is a key instrument used to measure apoptosis by several different parameters.

34. 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: (1995)
DNA is extracted from cells and run on a gel. When DNA is fragmented in cells undergoing apoptosis, the cleavage happens between nucleosomes - these are the structures of chromosomal DNA that ‘wraps’ around histones (represented like beads on a string) and there are approximately 180 nucleotides in each segment. The DNAases will cleave the DNA at sites where it is exposed, between the histone-containing nucleosomes.

35. FLOW CYTOMETER
A schematic of a flow cytometer, which has fluidics that draws in cells into a narrow flow of liquid so that the cells pass in a narrow stream, one cell at a time. The laser light can cause fluorescence of whatever type of ‘stain’ has been used to mark the cells. Detectors can detect light of various wavelengths and thus more than one parameter can be detected at the same time.

36. 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.
At the upper left, this shows a typical distribution of cells that are stained based on the binding of PI, which stains DNA. The biggest peak are cells with a single copy of DNA, in G1 phase of the cell cycle. As the cells duplicate their DNA in S phase, they accumulate more DNA, and then the second peak represents the cells that are in G2 and have twice as much DNA. In this analysis, the cells are fixed, which allows permeabilization of the membrane and the PI enters and stains the DNA. In the case of cells in which the DNA has been fragmented due to the cells undergoing apoptosis, the smaller fragments of DNA are allowed to leak out during the fixation process. These cells then have lesser amounts of DNA (‘sub-G1) and show up further to the left on the graph.

37. Use of TUNEL (TdT-Mediated dUTP Nick-End Labeling) to detect fragmented DNA in tissue sections
Normal Apoptosis induced

38. 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: , 2005.
In A and B, cells are stained with PI to detect DNA, but this dye only enters the cells when the membrane is disrupted, which occurs at late stages of apoptosis. Annexin V stains cells that are apoptotic because it is a protein that can bind to PS, which is externalized at the cell surface. Increasing cells detected in the upper left quadrant are those stained by Annexin V, but their membranes are intact. With time, more of these cells also stain with PI and show up in the upper right quadrant.
C and D shows that cells are stained based on higher caspase activity, and thus they appear further to the right (higher fluorescence).
Finally, stain of cells with a dye that binds to active caspases is another way to detect apoptotic cells.

39. References
Leber, B, Lin, J and Andrews, DW. Embedded together: The life and death consequences of the Bcl-2 family with membranes. Apoptosis 12, (2007).
Danial, NN and Korsmeyer,SJ. Cell death: critical conrol points. Cell 116, (2004).
Zimmerman, KC, Bonzon, Green, DR. The machinery of programmed cell death. Pharmacology and Therapeutics 92: (2001).
Duronio, V. The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem. J. 415: (2008).
Vandenabeele et al, Molecular mechanisms of necroptosis. Nature Rev. Cell Biol. 11, (2010).
Flow cytometry tutorial: