Transcript Document

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:
[email protected]
Web Page: http:/www.courseweb.uottawa.ca/BCH8109
Molecular mechanisms of neuronal death
• 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
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
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
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)
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.
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 9:3012:30 (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.
THE ASSESSMENT OF AN APPLICATION
The Primary Reviewer Report
1. 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).
2. 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.
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.
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?
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
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”.
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.
• Intros: Who are you? Who do you work for? What do you study?
• Assignment of Papers and Respondent Papers
• Break
• Assignment of Papers and Respondent Papers
• Break
The Basics: Neuronal Death by Design, Death By Default, Accidental
Death, and Autophagy
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Neurons
Nature Neuroscience - 10,
963 - 969 (2007)
Opposing roles in neurite
growth control by two sevenpass transmembrane
cadherins
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Yasuyuki Shima, Shin-ya
Kawaguchi, Kazuyoshi
Kosaka, Manabu Nakayama,
Mikio Hoshino, Yoichi
Nabeshima, Tomoo Hirano &
Tadashi Uemura
Astrocyte
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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)
Oligodendrocytes
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OPC migration and oligodendrocyte extension along a spinal cord axon
Nature Neuroscience - 9, 1506 - 1511 (2006)
Microglia
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Nature Neuroscience 8, 752 - 758 (2005)
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.
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.
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• 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
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 acute lack of
oxygen, elevated
temperature, contact
with toxic compounds,
excessive mechanical
strain (trauma).
Murder
Hallmarks: Cell explodes.
Cytoplasmic contents are
released eliciting an
inflammatory response
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
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
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.
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.
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
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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
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Nakagawa et al (2004) Autophagy Defends Cells Against Invading Group A
Streptococcus. Science 306:1037-1040
“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 (GFPtagged green) and were
killed upon fusion of these
compartments with
lysosomes. In autophagydeficient 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.”
The continuum of death: How can we come up with a
consensus that can be used to discuss cell death
Autophagy?
Apoptosis Apoptosislike PCD
Survival factors prevent
apoptosis within nutrientdeprived conditions
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Necrosislike PCD
Accidental
Necrosis/ Cell
Lysis
Classic Apoptosis: Chromatin condenses to
compact, globular, or crescent-shaped masses
at the nuclear periphery.
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
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
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)
•
Caspases are synthesized as (almost) enyzmatically inert
zymogens with 3 domains:
1. Prodomain
2. p20 domain
3. 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
Activation of caspases
1. Cleavage by an upstream caspase
Asp-X sites
•Mature enzyme is a heterotetramer (Two p20/p10
heterodimers with 2 active sites)
•Monomers may be active
2.
Aggregation is mediated by
adapter proteins
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
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
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)
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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.
Differences between pathways
Mitochondrial
(Death by default)
Death Domain
(Death by design)
-
-
Default pathway
Stress mediated
Requires new protein synthesis
Takes 12-24 hours to occur
Instructive apoptosis
Protein synthesis not required
(blocking protein synthesis
accelerates death)
-
Very rapid – death in a few hrs.
Slide courtesy of Dr. Ruth Slack, NRI, University of Ottawa,
http://www.ohri.ca/profiles/slack_summ.asp
Bad/Bax/BcL and death by default
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Molecular Biology of the Cell 4th edition
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
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CASPASEMEDIATED
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.
The continuum of death: How can we come up with a
consensus that can be used to discuss cell death
Apoptosis
Apoptosislike PCD
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Morphologically distinct
Caspase-dependent
Necrosislike PCD
Accidental
Necrosis/ Cell
Lysis
Peripheral chromatin condensation triggered by injection
of apoptosis-inducing factor (AIF). Caspase activation
was not required.
Apoptosis-like programmed cell death: Chromatin condensation is
less compact and “lumpier.” DNA fragmentation may not occur (i.e
no apoptotic ladders) and condensation may be incomplete Most
forms of caspase-independent cell death fall in this category (I.e
induced by AIF).
Morphological changes are not enough to determine
whether cell death is caspase-dependent or -independent.
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Zeiosis and apoptotic-like morphology in the absences of
caspase activation. Intensive zeiosis in Wehi-S murine
fibrosarcomas following death induction. Formation of a
terminal bleb marks cell death (energy failure) associated
with chromatin condensation. (Leist and Jaattela, 2003)
The continuum of death: How can we come up with a
consensus that can be used to discuss cell death
Apoptosis
Apoptosislike PCD
Necrosislike PCD
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Morphologically distinct
Caspase-dependent
Some but not all morphological
characteristics of apoptosis
Caspase-independent (not always)
Accidental
Necrosis/ Cell
Lysis
Multiple pathways triggered by death receptors
The continuum of death: How can we come up with a
consensus that can be used to discuss cell death
Apoptosis
Apoptosislike PCD
Necrosislike PCD
Accidental
Necrosis/ Cell
Lysis
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GIF decompres sor
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Morphologically distinct
Caspase-dependent
Some but not all
morphological
characteristics of
apoptosis
Fewer apoptotic characteristics,
mostly membrane associated with
little to no downstream DNAassociated changes
Caspase-independent Often ROS-mediated
(not always)
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Necrotic cell lysis triggered by 2 mM chloroquine
or oligomycin. A. Passive formation of a simple
terminal bleb (energy failure) in the absence of
zeiosis or cell movement. B. Cell lysis.
Is necrotic cell lysis really so simple?
The continuum of death: How can we come up with a
consensus that can be used to discuss cell death
Apoptosis
Apoptosislike PCD
Quick Time™ and a
GIF decompres sor
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Necrosislike PCD
Accidental
Necrosis/ Cell
Lysis
Energy and membrane
integrity failure
Little cell movement as
no cytoskeletal
restructuring
Morphologically
distinct
Caspase-dependent
Some but not all
morphological
characteristics of
apoptosis
Fewer apoptotic characteristics,
mostly membrane associated with
little to no downstream DNAassociated changes
Caspase-independent Often ROS-mediated
(not always)
Tying it all
together:
Ca2+ release
Calpain Activation
Cathepsin release
Curtain call:
Etoposide-induced apoptosis, apoptotic-like PCD, and necrotic-like
PCD in human neural progenitors (24 h treatment)
Tia Moffat - Ph.D 2006
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