Lecture 7 - Apoptosis

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First records: Chromatolysis…

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Apoptosis history

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In the 1960s it was observed that ischaemic liver tissue showed scattered hepatocytes in surviving parenchyma, with small masses of cytoplasm and specks of condensed nuclear chromatin
The name shrinkage necrosis was suggested (Kerr, 1971)

Early in the 70s Currie, Wyllie and Kerr detected shrinkage necrosis in a set of other experiments, and it became clear that this is a distinctive form of cell death phenomenon with homeostatic function on cell population size (Kerr et al., 1972)

It was decided to name it apoptosis, a morphological term from the Greek “falling off”, as autumn leaves fall off trees in an inherently seasonal manner

Phenomenon called programmed cell death received its name before apoptosis (Saunders, 1967, Lockshin, 1971)

it referred to situations in which cells are programmed to die at a fixed time (death on schedule – eg of clusters of cells in the embryo)

These doomed cells die on schedule even if they are transplanted elsewhere in the embryo

In most (but not all !) cases the morphology of this death on schedule turns out to be apoptosis

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Apoptosis is a process with distinct features

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In 1976 and 1981

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electrophoretically the chromatin of irradiated tissues broke down into fragments that produced a typical, ladder-like pattern

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Wyllie et al in 1984

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linked the ladder pattern with the phenomenon of apoptosis and thereby added a specific biochemical marker to the distinctive morphological changes

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Apoptosis is a form of cell death characterized by

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morphological as well as biochemical criteria (and both must be carefully utilized)

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Morphologically:

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cell shrinks and becomes
denser (shrinkage necrosis), chromatin becomes pyknotic and packed into smooth masses against the nuclear membrane (margination of chromatin)

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Apoptosis may be separated into phases of

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initiation or induction, integration or determination and execution

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Apoptosis is a process with distinct features
The process:

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The nucleus may break up (karyorhexis)
cell emits processes (budding phenomenon) that often contain pyknotic nuclear fragments (in vitro vs in vivo!)
processes tend to break off and become apoptotic bodies, which may be phagocytized by macrophages
little or no swelling of mitochondria or other organelles, cell membrane remains intact (until very late)
DNA is broken down into segments (multiples of approximately 185 bp), due to specific cleavage between nucleosomes
The process is under genetic control and can be initiated by an internal clock (development), or by extracellular agents such as hormones, cytokines, killer cells and a variety of chemical, physical, and viral agents
Role of microenvironment driving genetic control mechanism

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signal for apoptosis can either arise from

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the extracellular or intracellular environment, giving rise to an intrinsic and extrinsic apoptotic pathway

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Extrinsic apoptosis

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Apoptosis can be initiated by extracellular molecules that bind to ‘death’ receptors (for example, FAS), in turn activating the caspase 8– caspase 3 cascade

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extrinsic pathway relies on

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extracellular death ligands, which transmit the signal via caspases within the cytoplasm, which ultimately activate the same executioner caspases as employed by the intrinsic pathway

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Intrinsic apoptosis

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Intracellular stress is sensed by mitochondria, which undergo mitochondrial membrane permeabilization and hence activate caspase-dependent and caspase-independent cell death executioner mechanisms

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In Intrinsic apoptosis, which factors control mitochondrial membrane integrity

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Pro- vs anti-apoptotic factors control mitochondrial membrane integrity

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Mitochondria:

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sequestering pro-apoptotic signals and releasing cytochrome c, required for cytosolic caspase-9 activation

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Both extrinsic and intrinsic pathways

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merge on the level of execution with caspase-3

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Fluorescence microscopy and flow cytometry:

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single cell and population level assessment

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Capase-3 and its downstream target PARP

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Caspase 3 becomes activated through cleavage, seen in a decrease in band signal, as its cleaved counterparts migrate further in the gel
Caspase 3 in turn cleaves PARP, a DNA repair enzyme

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Dynamics within modes of cell death
Defining the PONR

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Existence of the point-of-no-return (PONR) has been suggested
The PONR: distinction of cell death induction from cell death execution, cell death as a process from cell death as endpoint and thus the distinction between a living and an already dead cell
Onset delay = inhibition

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PONR gives a

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Better understanding of the time point when a cell is still salvageable

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Suggested events defining PONR:

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Mitochondrial membrane permeabilization
Caspase activation
Dissipation of mitochondrial transmembrane potential ∆Ψm

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only a dynamic perspective on the position of the PONR can reveal its significance

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WHat are good molecular markers that can be used for apoptosis (make some notes)