BCH 8109 / MIC 8123 ADVANCED TOPICS IN CELL DEATH (3 cr.) Tuesdays, 9:30-12:30, Rm 4161 RGN Coordinator: Dr. Steffa

1. BCH 8109 / MIC 8123 ADVANCED TOPICS IN CELL DEATH (3 cr.) Tuesdays, 9:30-12:30, Rm 4161 RGN Coordinator: Dr. Steffany Bennett Office: Rm 4261 RGN Email: sbennet@uottawa.ca Web Page: http:/www.courseweb.uottawa.ca/BCH8109 Molecular mechanisms of neuronal death

2. Course Description: Expectations, on-line documentation, questions • Intros: Who are you? Who do you work for? What do you study? • Assignment of Papers and Respondent Papers • The Basics: Neuronal Death by Design, Death By Default, Accidental Death, and Autophagy

3. Evaluation: • 1. 30% Class Presentation • (30 minute presentation/ 15 min question period) • (20 marks from Dr. Bennett, 10 marks from colleagues) • Presentation of a paper assigned by one of the lecturing professors. Presentation should be prepared using PowerPoint or other software supported by University of Ottawa Medtech services. A data projector, laptop, with CD and Zip drive will be available • Marking scheme • 10 marks • presentation of background material necessary to understand the hypothesis • presentation of introduction, results, methods • 10 marks • critical analysis of discussion • - presentation of controversies inherent in the research • - ability to promote discussion and questions from student colleagues

4. 2) 10% Respondent • Marking scheme • 10 marks • each student will act as a ‘respondent’ for one student presentation. The respondent will be required to be familiar with the article prior to the presentation and open the question period following the student presentation. • - the grade will reflect how well the respondent initiates discussion and/or offers alternative explanations or critique of the paper being discussed

5. 3) 10% Class participation • Marking scheme • 10 marks • participation in class discussions and presentations • students will be asked to hand in one question per paper under discussion at the beginning of each class. The lecturer and coordinator will evaluate the depth of the question and assign a mark of 0, 0.5 or 1 mark • students may miss one class without penalty. The grant panel is mandatory as participation affects not only your own mark but that of your colleagues • students will be asked to grade the presentations and provide their colleagues with feedback using a marking scheme provided at each class. • the class participation mark will be an combination of the student evaluations (1/2 of grade) and the evaluation marks assigned by the course coordinator (1/2 of grade)

6. 4) 30% BCH8109 Grant Proposal Students are required to identify a “potential supervisor” working on an aspect of cell death of interest from their examination of the literature. Students will write a 6 page post-doctoral research proposal to work in this supervisor’s laboratory using the forms and guidelines posted on the class web-site. Grants will be subjected to a peer review process based on the CIHR peer review system. 50% of the grade for the grant proposal will be determined by this review process (15 marks). The top grant as determined by peer review will be “funded.” The remaining 50% of the grade for the grant proposal will be determined by the course coordinator (15 marks). GRANT PROPOSALS ARE DUE NOV 27 AT THE BEGINNING OF CLASS. Proposals not handed in at the beginning of class will receive a mark deduction.

7. 5) 20% Grant Review (10 marks) and Participation in Grant Panel (10 marks) Grant Review: Students will be assigned two proposals written by colleagues in the class. All proposals will be identified by student number only (anonymous). Each student will act as a primary reviewer of one of the proposals and a secondary reviewer of the other. Students will write a 2 pg review of the proposal for which they are the primary reviewer. This written report will make up 50% of the mark for this section and will be graded by the course coordinator (10 marks). Reviewers will receive their grants in class on Dec 4. Grant reviews are due at the grant panel Dec 11. Grant panel will be held Dec 11 9:30-12:30 (Panel A) and 1:30 to 4:30 (Panel B) Nov 13: For those who wish to participate in the grant writing workshop (Optional), a draft of your summary page of your grant proposal must be handed in at the beginning of class. This summary should be anonymous.

8. THE ASSESSMENT OF AN APPLICATION The Primary Reviewer Report The individual review should briefly outline the aim(s) and description of the project - purpose of the proposal, the hypothesis to be tested or question to be addressed, the objectives to be achieved, the approach proposed, and the progress made to date (published data generated in the “supervisor’s laboratory included by the grant applicant as figures). This is followed by an assessment of the application (see next slide). It should be clear and concise, using objective and non-inflammatory language. The reviewer should state his/her opinions, with justification.

9. Assessment of the Application: The review should contain an assessment of the strengths and weaknesses, the originality and potential impact of the research described in the application, and the appropriateness of the research plan. The most useful reviews are those which provide constructive advice to the applicant, enabling him/her to improve the quality and efficiency of the proposed research. Criteria are provided on the next slide.

10. Assessment criteria: * How important and/or original are the hypotheses or the questions to be addressed, and how clearly are they formulated? * How important and original are the contributions expected from the research proposed? What is the potential for important new observations or knowledge that will have an impact on the health of Canadians? * How well will the proposed experiments address the hypotheses or questions? How appropriate are the methods to be applied and the proposed analyses of data? How well will the applicant implement new methods which are to be introduced and/or explored? How well have the applicants anticipated difficulties in their approach and considered alternatives? * Is the rationale for the proposal well-grounded in a critical review of the pertinent literature?

11. At the end of the report, the primary reviewer will assign a score according to the following rating scale: 4.5 - 5.0 outstanding 4.0 - 4.4 excellent 3.5 - 3.9 very good 3.0 - 3.4 acceptable, but low priority 2.5 - 2.9 needs revision 2.0 - 2.4 needs major revision 1.0 - 1.9 seriously flawed

12. 5) 20% Grant Review (10 marks) and Participation in Grant Panel (10 marks) Participation in grant panel: Dec 11 will be a peer review grant panel. The class will be divided into two panels. Panel 1 will meet 9:30-12:30. Panel 2 will meet 1:30-4:30. Presentation of the grant review, participation as secondary reviewer, and panel participation will make up the remaining 50% of the mark for the grant review and participation section (10 marks) Each student will be responsible for presenting the proposal they reviewed as primary reviewer to the panel. The secondary reviewer will be asked to make additional comments on the grant. Both reviewers must give their score to the panel after the review. The panel will discuss the proposal, asking the primary and secondary reviewers for further clarification where needed. Each member will provide a score out of 5. The average score will be the grant “rating”.

13. The application receiving the top grant rating as determined by peer review will be funded. Panel results will be available on-line by Dec 18th. Final marks will be posted as soon as possible after Dec 18th.

14. Intros: Who are you? Who do you work for? What do you study? • Assignment of Papers and Respondent Papers • Break

15. Assignment of Papers and Respondent Papers • Break

16. The Basics: Neuronal Death by Design, Death By Default, Accidental Death, and Autophagy

18. Neurons Nature Neuroscience - 10, 963 - 969 (2007) Opposing roles in neurite growth control by two seven-pass transmembrane cadherins Yasuyuki Shima, Shin-ya Kawaguchi, Kazuyoshi Kosaka, Manabu Nakayama, Mikio Hoshino, Yoichi Nabeshima, Tomoo Hirano & Tadashi Uemura

19. Astrocyte Two-photon transmitted and Ca2+ indicator fluorescence overlay showing arteriole constriction restricted to the region adjacent to the Ca2+ rise in the single astrocyte endfoot resulting from a Ca2+ wave initiated out of frame (same experiment as in Figure 2 D-F). Acquisition rate = 3.1 sec/frame (61 frames) http://www.neuropathologyweb.org/chapter1/chapter1bAstrocytes.html Calcium transients in astrocyte endfeet cause cerebrovascular constrictions Nature 431, 195-199(9 September 2004)

20. Oligodendrocytes OPC migration and oligodendrocyte extension along a spinal cord axon Nature Neuroscience - 9, 1506 - 1511 (2006)

21. Microglia Baseline dynamics of fine microglial processes. Microglia in the cerebral cortex display a highly branched morphology with each cell soma decorated by long processes with fine termini. Timelapse imaging of microglia in the intact mouse brain reveals rapid extension, retraction, shape and volume changes of fine processes over intervals of seconds to minutes, while microglial cell bodies and main branches remain morphologically stable over hours. Nature Neuroscience 8, 752 - 758 (2005)

22. Rapid microglial response after laser-induced ablation. • a small laser ablation is induced ~40 µm from the pial surface. • Within the first minutes post-ablation, the tips of the processes of the cells immediately surrounding the ablation appear bulbous and slightly enlarged. • In the next few minutes, these cells extend their processes toward the damaged site where they appear to fuse together and form a spherical containment around it. • During this period, the same cells also retract those processes in directions opposite to the site of injury. • Most of the cellular content of each of the immediate neighbors is directed towards the damaged site within the first 1-3 hours, whereas the cell bodies remain at approximately the same location for at least 10 hours

23. Apoptosis vs Autophagy vs Necrosis: Three deaths or one continuum If cellular homeostasis is severely compromised, a cell cannot maintain a stable internal environment and is lysed. Environmental conditions that trigger necrosis include acutelack of oxygen, elevated temperature, contact with toxic compounds, excessive mechanical strain (trauma). Murder Hallmarks: Cell explodes. Cytoplasmic contents are released eliciting an inflammatory response

24. Apoptosis vs Autophagy vs Necrosis: Three deaths or one continuum Cells are genetically programmed to initiate cell death upon a) exposure to a death ligand (death by design) or b) removal of factors required for cell survival (death by default). Suicide Hallmarks: Cell is disassembled through a series of orderly events orchestrated by gene expression

25. Apoptosis vs Autophagy vs Necrosis: Three deaths or one continuum When cells are no longer exposed to sufficient nutrients in their microenvironment, they can cannibalize some of their internal organelles such as the mitochondria to re-use these components. A catabolic process by which cells degrade and digest their own cytoplasmic constituents, usually through the action of lysosomal enzymes. One of the most distinguishing features of autophagy is the dynamic rearrangement of cellular membrane to sequester cytosol and organelles into autophagosomes for delivery to the lysosome or vacuole. Self-cannibalism Autophagy Hallmarks: Formation of a double membrane within the cell which envelops the materials to be degraded into a vesicle called an autophagosome. The autophagosome then fuses with a lysosome whose hydrolytic enzymes degrade the materials

26. Membrane breaks down resulting in an inflammatory response Edema Membrane intact until phagocytosed Pyknosis The term apoptosis was first defined by Kerr, Wyllie, and Currie (1972) Br. J. Cancer 26: 239-257 based on morphological criteria.

27. Macroautophagy. Formation of a double membrane within the cell which envelops the materials to be degraded into a vesicle called an autophagosome. The autophagosome then fuses with a lysosome whose hydrolytic enzymes degrade the materials.

28. Note: Cell was irradiated ~ 50 h before death. 46.5 h after irradiation necrotic transition was observed. Note that it took less than 10 min for cell membrane to rupture and explode (C-D) http://www.ucsf.edu/cvtl/prev/necrosis.html

30. Note: Cell was irradiated ~ 29 h before death. 14.5 h after irradiation apoptotic morphology was observed. Note that it took approximately 20 min from morphological change to death Irradiated with 4 Gy http://www.ucsf.edu.cvtl/prev/apoptosis1.html

32. “We found that the autophagic machinery could effectively eliminate pathogenic group A Streptococcus (GAS) (red) within nonphagocytic cells. After escaping from endosomes into the cytoplasm, GAS became enveloped by autophagosome-like compartments (GFP-tagged green) and were killed upon fusion of these compartments with lysosomes. In autophagy-deficient Atg5–/– cells, GAS survived, multiplied, and were released from the cells. Thus, the autophagic machinery can act as an innate defense system against invading pathogens.” Nakagawa et al (2004) Autophagy Defends Cells Against Invading Group A Streptococcus. Science 306:1037-1040

33. The continuum of death: How can we come up with a consensus that can be used to discuss cell death Autophagy? Necrosis-like PCD Apoptosis-like PCD Accidental Necrosis/ Cell Lysis Apoptosis Survival factors prevent apoptosis within nutrient-deprived conditions

35. Classic Apoptosis: Chromatin condenses to compact, globular, or crescent-shaped masses at the nuclear periphery.

36. Caspases elicit morphological change • Cytosolic (or Cysteine-dependent) Aspartate-Specific Proteases CASPases • Cysteine proteases • Main effectors of apoptosis - elicit most of the morphological changes observed in apoptotic cells by cleaving a wide variety of substrates (Exceutioners). Initiator caspases activate executioner caspases. • Large protein family, highly homologous • Highly conserved through evolution - humans to nematodes • At least 13 caspases have been identified in humans - 2/3 of these proteins are implicated in apoptosis

37. Caspase cleavage • All known caspases possess an active-site cysteine (conserved motif QACXG) • Substrates are cleaved at Asp-Xxx bonds (ie after aspartic residues) • Caspase substrate specificity is determined by the four AA residues that are N-terminal to the cleavage site *** • Caspases are divided into subfamilies based on their substrate preference, extent of sequence identity, and structural similarities

38. Caspases as the central executioners of apoptosis • Proteolysis brings about most of the visible changes that characterize apoptotic death • Blocking caspase activity (mutations or inhibitors) delay and, in some cases, prevents apoptosis and thus rescues dying cells (simplistic)

40. Caspases are synthesized as (almost) enyzmatically inert zymogens with 3 domains: • Prodomain • p20 domain • p10 domain • Caspases are activated by proteolytic cleavage of the zyomogen between the p20 and p10 domains and often between the prodomain and p20 domain

41. Asp-X sites Activation of caspases 1. Cleavage by an upstream caspase • Mature enzyme is a heterotetramer (Two p20/p10 heterodimers with 2 active sites) • Monomers may be active

42. Caspase 8 is a key initiator caspase in “death by design”. If multiple caspase 8 molecules aggregate then the basal protease activity of the parent zymogen may be sufficient to mutually cleave and activate each other Aggregation is mediated by adapter proteins 2.

43. 3. Holoenzyme formation • Caspase 9: Complex activation mechanism • Cleavage has only a minor effect on activity • Key requirement is association with a dedicated protein cofactor Apaf-1 • Holoenzyme formation is induced by oligomerization of Apaf-1 (dependent upon ATP and cytochrome c (released from mitochondria)) • Oligomerization permits recruitment of caspase 9 into the apoptosome complex • The conformational change elicited in caspase 9 by becoming part of the apoptosome complex results in activity

44. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant J C Goldstein, N J Waterhouse, P Juin, G I Evan & D R Green Nature Cell Biology 2, pp 156 - 162 (2000) This cell expresses cytochrome c fused to the green fluorescent protein (GFP). After treatment, cytochrome c-GFP (green)moves from the mitochondria to the cytosol.

46. Mitochondrial (Death by default) Default pathway Stress mediated Requires new protein synthesis Takes 12-24 hours to occur Death Domain (Death by design) Instructive apoptosis Protein synthesis not required (blocking protein synthesis accelerates death) Very rapid – death in a few hrs. Differences between pathways Slide courtesy of Dr. Ruth Slack, NRI, University of Ottawa, http://www.ohri.ca/profiles/slack_summ.asp

47. Bad/Bax/BcL and death by default Molecular Biology of the Cell 4th edition

48. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant J C Goldstein, N J Waterhouse, P Juin, G I Evan & D R Green Nature Cell Biology 2, pp 156 - 162 (2000) PUTTING IT ALL TOGETHER CASPASE-MEDIATED APOPTOSIS This cell expresses cytochrome c fused to the green fluorescent protein (GFP). After treatment, cytochrome c-GFP (green)moves from the mitochondria to the cytosol. The cell then rounds, blebs, and externalises phosphatidylserine, a phospholipid membrane component and phagocytosis signal identified by association with annexin V (red). Finally a DNA dye (blue) stains the nucleus following the collapse of plasma membrane integrity. The interval between each frame is ten minutes.

49. The continuum of death: How can we come up with a consensus that can be used to discuss cell death Necrosis-like PCD Apoptosis-like PCD Accidental Necrosis/ Cell Lysis Apoptosis Morphologically distinct Caspase-dependent