Cell Death and Neurotrophic factors Flashcards
how can cells undergoing apoptosis be distinguished?
their nucleus becomes very dark and enlarged as the chromatin condenses and cytoplasmic blebbing
what are the two ways in which cell death can occur
necrosis or apoptosis
how is apoptosis carried out?
activated of caspases which are proteolytic enzymes that break down the nuclear lamina and cytoskeleton so that the cell can be cleared safely by macrophages- the DNA is also fragmented
describe the process of apoptosis molecularly
- there is an influx of ions into the mitochondria
- release of cytochrome C which binds to Apaf 1
- activation of Caspase 9
- Activation of Caspase 3 via cleavage
- caspase 3 degrades cellular components
what things normally stimulate cell death?
- a lack of growth factors
- stimulation of apoptosis pathway
what happens when you KO caspase 9 in the mouse?
you get an overgrown brain
what receptor normally stimulates apoptosis when bound?
Fas or TNFH
describe an experiment which looked at the effect of removing or adding a limb bud to an embryo… what did this show?
if can use a strain such as DiI which will stain the entire neuron. Normally you see that the motor neurons are all present in the ventral part of the cord. You can then use one limb as a control and the one on the opposite side to experiment. You can remove a limb bud and you see that the motor neuron number decreases. you can also add a limb bud and you will see that you get more motor neurons in the ventral region.
- This shows that the number of motor neurons is not set and can vary depending on the presence or absence of a limb- maybe the limb sends a signal
how was it shown that cell death of neurons varies according to whether along the spine the DRG is? what happens when you then ablate a limb
if you measure the DRG death rate in neurons you find that there is less death in the DRG that innervate limb buds that in other regions. This suggests that “all DRG neurons are created equally” and cell death occurs depending on where the DGR is situated. This is further supported by the fact that if you ablate a limb then the segment gets just as much cell death as the non limb DRGS- so the death rate is depending on the limb
what is the neurotrophic hypothesis?
this is the hypothesis that neurons compete for a limited amount of neurotrophic factor that is produced by their target. Those neurons that receive sufficient ‘trophic support’ survive and others die. Survival requires a retrograde signal from the periphery to the cell body
describe how the NGF was discovered.
- they implanted tumour cells into the limb bud. They found that this promoted the survival of sensory and sympathetic neurones. There was a huge growth in the ganglia. This showed that there was something in the tumour that could promote survival.
- they then placed tumour cells on on the chorioallantoic membrane promoted survival of sensory and sympathetic neurons. This shows that the sarcoma -derived surival factor is diffusible!
- they then cultured DRG on the same culture that a mouse sarcoma was cultured on and saw it promoted growth
- for some reason they thought it was nucleic acid and they knew that snake snake venom phosphodiesterase could break down nucleotides so they added this- saw that it still grew so then they tried venom alone and it worked
- they purified and found NGF!
how did they investigate whether NGF promoted growth or just survival?
they used antibodies against NGF and found that there was reduced growth and cell survival in the DRG cultured
how did they investigate that NGF was a ligand?
they knew that a good ligand has a low effect at low levels and a high affect at high levels so they did this with NGF and looked at the survival rate and found it was dose dependent and plateaued.
how did they show that NGF worked via retrograde signalling?
they used a culture chamber that allows neuronal cell bodies to be exposed to different environments. they put NGF with the peripheral sons and nothing in the cell body chamber- the survival and growth was improved- not when you put antibody in
how did they test whether NGF also works on type of neurons other than motor neurons? what did this mean?
they carried out the same culture experiments with sympathetic neurons and sensory neurons but not on any others including spinal motor neurons- this means that NGF can’t be the whole story, there must be more
