Lecture 30- Neurotrophins I (Apoptosis) Flashcards
What was the discovery made by Zacharias Janssen around the year 1595 on the field of microscopy?
•the 1590s •eyeglasses were beginning to be widely used •Dutch spectacle makers •experimented with two lenses in a tube – things look a lot bigger •‘micro’ means ‘small’, ‘scope’ means ‘to see’ •Magnified 10 to 20 times
What was the discovery made by Robert Hooke around the year 1665 on the field of microscopy?
•devised the compound microscope •In 1665 published ‘Micrographia’ •was the first to use the word “cell” •described the smallest unit of a living organism
What was the discovery made by Anton van Leeuwenhoek around the year 1674 on the field of microscopy?
•made microscopes as a hobby •grinding and polishing of lenses •achieved magnification of 200 to 300 times •discovered bacteria and protozoa
What is the cell theory that was discovered in the 1700s and improved on in 1838?
– ‘The cell is the fundamental element of organization’ - All life forms are made from one or more cells - Cells only arise from pre-existing cells - The cell is the smallest form of life - this theory was made possible only by advances in microscopy, and is now one of the foundations of biology 
What is the brief history of cells and cell death?
1: 1838: the Cell Theory 2: 1842: Carl Vogt - Zoologist, studying tadpole development - describes the principles of cell death 3: 1896: John Beard - describes “the programmed loss of an entire population of neurons in fish embryos.” 4. 1965: John Foxton Ross Kerr - at University of Queensland - studied liver tissue using electron microscopy - morphologically distinguished apoptosis from traumatic cell death (difference between programmed cell death and injury induced death)
What is apoptosis?
- ‘apo’ from/off + ‘ptosis’ to fall - the process of programmed cell death (PCD) - discrete biochemical events and characteristic cell morphology changes -between 50 and 70 billion cells die each day due to apoptosis in the average human adult -vacuoles bud off, then membranes surround the organelles and those get consumed by macrophages= ordered way to die for a cell
What is necrosis?
- ‘dead’ - traumatic, a result from acute cellular injury - inflammatory changes -trauma, swell, leak and spill their insides to the surroundings, that triggers inflammatory response that can be bad
What happens in apoptosis?
- Often happens to healthy cells
- Stereotyped process
Cells removed with minimal disruption:
- pyknosis
- DNA cleavage
- cytoplasmic blebbing
- phagocytosis
- clean, efficient and normal part of development
What happens in necrosis?
- Only happens to sick or damaged cells Involves local inflammation
- swelling
- lysosomal activity
- cell lysis & debris
- inflammatory response
Is the machinery of apoptosis similar in all cells?
-yes -biochemically and morphologically stereotyped -requires mRNA and protein synthesis -requires mRNA and protein synthesis -apoptosis is an active process
Does every cell have the machinery for apoptosis?
-yes -thus apoptosis must be tightly regulated -initiated only on specific signals in damaged/stressed/unwanted cells
What are the types of triggers that trigger apoptosis?
-extrinsic signals (eg. Fas ligand) -intrinsic signals (eg. cytochrome C) -trigger can include extrinsic signals (another cell signals it to die) -intrinsic signal (suicide, realizes that it is in the wrong place)
What is the extrinsic pathway that can trigger apoptosis?
- family of receptors TNFR (tumor necrosis…) and have Fas ligands
- this was discovered by looking at virus infected cells
- Fas binds, form clustering at the receptor (bind as a trimer) and that activates signals -signals that induce= it is apoptosis, signal the cell to die!
- Fas ligand-receptor activation
- member of the Tumor Necrosis Factor Receptor Superfamily Causes apoptosis in virus-infected cells:
- Fas ligand produced by lymphocytes
- Fas receptor expressed on target cell
- Ligand clusters the receptors attracts adaptor proteins and concentrates apoptosis initiators
What are the steps in extrinsic controls triggering apoptosis?
- receptor clustering
- ‘death domain’ aggregation
- recruitment of adaptor proteins (eg. FADD)
- facilitates binding of caspase 8 -caspases: cysteine-requiring aspartate proteases
- large protein family -inactive procaspases, activated by cleavage
- cleave proteins at aspartic acid residues - forms the death-inducing signaling complex (DISC)
- acts as ‘initiator’ caspase; cleaves other ‘effector’ caspases (so will have more types of caspases active)
- ‘cascade’ effect follows
- cleave nuclear laminins, breakdown nuclear lamina, activate DNAse, cell apoptosis (caspases= will cleave other caspases but also start cleaving DNA, pretty much dismantling the cell)
What do caspases exist as normally?
-caspases exist as proforms, inactive, wait for a signals= the clustering etc is what activated -the prodomain gets cleaved off= then have an activa caspase molecule =the caspase 8
What is the intrinsic control of apoptosis?
- Intrinsic mechanism of death:
- mitochodria will function fine when cell is in the right environment, cytochrome C in the mitochodria if cell healthy
- if not right signals= leak cytochrome C which triggers apoptosis
- Bcl2 family control the cytochrome C (don’t have to know the individual Bac names, only Bcl2)
- some of the family is pro apoptotic, some anti= have an equilibrium if healthy
- then upset the balance= and cytochrome leaks= apoptosis
What is the intrinsic signal for apoptosis?
-Signal is an increase in cytoplasmic Cytochrome Complex (Cytochrome C) - cytochrome c normally in mitochondria (electron transport chain) - leakage of cytochrome c into cytoplasm triggers apoptosis
What is the family of proteins regulating the cytochrome c leakage?
-Regulated by the Bcl2 family of proteins - pro-apoptotic: Bax, Bak, Bad, Bim, Bid - anti-apoptotic: Bcl2, Bcl-XL - block or promote pore formation
What are the steps in intrinsic control of apoptosis?
- release of cytochrome C
- cytochrome C binds to adaptor protein Apaf-1
- recruitment of procaspase 9
- formation of apoptasome
- activation of ‘executioner’ caspases
- ‘cascade’effectfollows
- cleave nuclear lamins, breakdown nuclear lamina, activate DNAse, cell apoptosis
What is the intrinsic pathway controlled by?
-Intrinsic pathway controlled by ‘growth factor’ signals - usually prevents apoptosis - absence of signal leads to apoptosis - ‘growth factor’ signaling affects multiple pathways -growth factors are crucial= maintain the Bcl2 members, if have the right growth factors= have antiapoptotic ones, if move the cell and different growth factors= then have pto apoptotic
What happens when the growth factors fail?
-failure of growth factors signal= pro apoptotic Bcl2 mebers break up the anti apoptotic ones - cytochrome C leaks via pores that form= then bind to aph1= form an apotome and then cleave everything and cell dies
How can the extrinsic and intrinsic pathway interact?
-killer cell will have the TNF ligand = recruits a number of receptors -caspase is activated= when have 3 normally, then activate each other, cleave the domain and can activate other caspases -can also activate the intrinsic pathway and get cytochrome c leakage and get apoptasome= that can activate more caspases and cleavage happens and cell dismantled -caspases break the structural elements of the cell
What is the comparison and interaction of the extrinsic and intrinsic pathways?
- pathways intersect:
1. Extrinsic:
- direct activation of caspases
- activation of intrinsic pathway via pro-apoptotic Bcl2 members
2. Intrinsic:
- activation of caspases via cytochrome C
- Both pathways converge on caspase activation
- 2 broad pathways: the extrinsic one can activate the intrinsic -the intrinsic can work independently, dependant on the health of the cell
Are the apoptotic pathways conserved across species?
- Key molecules in the apoptotic pathway are highly conserved through evolution
- diversity in how cells have evolved to execute themselves but a conserved mechanism, still caspases and Bcl2 family involved
- Genetics has confirmed the key roles they play in this process
Are the apoptotic pathways complex?
-Apoptotic Pathways: considerably more complex -Evolutionary expansion of receptors containing “death domains” -Cell specific driven expression of receptors and adaptor proteins -Context-driven signaling: the outcome is not always cell death -the pathways are more complex, the cell will always have one of the TNF receptors, many types -this is one of the way neurotrophins control cell survival via p75 -there is context, TNF signalling doesn’t always result in death
How does the expression of genes change during apoptosis?
- don’t have to know details of the experiment
- the lines represent RNA (blue= low expression, red= high expression)
- which genes are upregulated in the apoptosis process
- the red ones= clusters of genes that control the apoptotic process!
- need clusters of genes for apoptosis
What techniques are used to detect apoptosis?
-Common methods for measuring Apoptosis takes advantage of the properties of dying cells: 1. annexin V staining 2. TUNEL staining 3. Caspase 3 staining 4. Morphological features
What happens when you knock out the Apaf-1 in mice?
-Disruption of brain development by blocking apoptosis -‘knock-out’ of caspase-9 or Apaf-1 in mice -normal neural apoptosis fails to occur -over generation of brain neurons is obvious -enlarged brain protrudes above the face -knocking out aph-1= knock outs normal cell death= way too big head -can take the genetic approach= delete Bcl2, Aph1, caspase 9 etc.
What is the general principle of neuronal development?
- many neurons born during development die soon after - morphologically observed to die by apoptosis - quantified by counting, but very difficult to estimate • 0-80% of neuronal populations die • often overlaps with period of cell division • cells change size as they grow • glial cells can also die –normally more neurons formed then we need -no single rule for all populations, in some none die
What did Viktor Hamburger do?
- embryologist who focused on neural development - removal of peripheral target was known to prevent development of innervating neurons -cut off a limb bud in a chicken embryo -then get one legged chick -look at the spinal cord= lose the motor neurons -interpretation: the limb bud regulates the number of neurons - Concluded that the target regulated neuronal number, controlled proliferation and differentiation of neuroblasts Ultimately led to “The Neurotrophic Hypothesis” of neuronal survival
