Lecture 31- Neurotrophins II Flashcards
What were the experiments of Viktor Hamburger?
- removal of peripheral target was known to prevent development of innervating neurons
- cut off a limb bud=then fewer neurons on that side
- also adding a limb bud to a chick, so have side with 2 legs, then that side of the spinal cord= more motor neurons
- so he thought that sth in the limb bud increases the proliferation of neuroblasts
- Concluded that the target:
- regulated neuronal number
- controlled proliferation and differentiation of neuroblasts
What did Rita Levi-Montalcini do?
• Jewish-Italian scientist in Fascist Italy • Repeated Hamburger’s experiments: similar experiments to Hamburger, slightly different to his results - nerve cells did not wither immediately - neurons proliferated, differentiated and grew towards target - the problem was the lack of a survival-promoting factor
What did the initial experiments of Rita Levi-Montalcini lead to?
-The neurotrophic factor hypothesis: -Targets of innervation secrete limiting amounts of survival factors to generate a balance between the size of the target organ and the number of innervating neurons.
What do the examinations neuronal cell death reveal (Viktor Hamburger)?
- counting the number of neurons in the spinal cord
- there is a critical period when the motor neurons would die the the absence of trophic support or when the limb bud wasn’t there, this was when they were reaching the target
- Neurons go through a ‘critical period’ where apoptosis occurs in the absence of trophic support
- In many cases this coincides when neurons innervate their target tissue
- The number of degenerating neurons peaks when the loss of viable neurons is most rapid
What is the pattern of naturally-occurring cell death in chick dorsal root ganglia?
- less cell death in the DRGs that innervate limb buds (blue filled)
- the size of limb DRGs decrease only slightly during development
- the size of axial DRGs become much smaller
- neuron survival is correlated with the amount of target tissue DRG= dorsal root ganglia
- the DRG that wouldn’t innervate limbs would lost more cells than the ones that would
- neuronal size and survival is correlated with amount of target tissue
What was the experiment with the mouse tumour and chick embryous?
-A target-derived soluble factor can support neuron survival – a tumor cell line placed in the chick embryo at E3 - the nerve fibers have direct access - SGC and DRGs are much larger ipsilateral to tumor - same tumor cell line is placed in the yolk sack - the nerve fibers have no direct access - All SCG and DRG are increased in size -The tumor must have secreted a soluble factor that enhanced neuron survival -Experiment 1:put a bit of the tumour from the mice, and put into chick, then more neurons innervating the tumours -the tumours was producing a growth factor for neurons -2nd experiment: put tumour into yolk sac so not in contact with tissue of the embryo, here more neurons in the whole embryo - chick embryonic DRG neurons - mouse sarcoma conditioned medium added - haloes of nerve fibres grew from the DRG, with their highest density facing the tumour - A soluble factor that supports the survival and growth of DRG neurons is discovered in a mouse sarcoma -Then did in vitro assays: -if put the tumour the DRG produced many neurites, the neurites would grow mostly towards the tumour, even directing the neurites -hypothesis: nucleic acid?
What was the experiment with the mouse tumour and snake venom?
- testing if it is a nucleic acid the growth factor: -testing it via snake venom as it is known to destroy nucleic acid
- Neurite outgrowth assay:
- sarcoma conditioned medium added - snake venom added to the culture = DRG neurons survived and grew
- When only snake venom was added, a surprising discovery was made: the DRG neurons survived and grew
- The snake venom must also have contained a soluble survival factor (increased survival of neurons in both the tumour and venom, and venom only!!!)
How was the nerve growth factor (NGF) identified and purified?
-Venom gland is a salivary gland -Mouse salivary gland subsequently shown to contain abundant neurotrophic activity -now took cow salivary glands= the salivary glands and they also increase survival= neurotrophic activity -also with mouse salivary glands -then could purify the growth factor: from the mice salivary glands -now that they had pure NGF could do new experiments: -1960: Protein purified from mouse salivary gland ‘Nerve Growth Factor’ -1969: NGF purified to homogeneity -Research tools developed: -purified NGF -activity blocking antibodies -measurement assays via radiolabelling
What does the NGF look like?
-then NGF was sequenced in several species: the purple that is conserved across species, extremely conserved across species -Neurotrophins are generated as larger proteins and then cleaved to become active - synthesized as a preproprotein of ~305 amino acids - ‘mature’ NGF dimer obtained by proteolytic processing of the precursor protein - ~118 amino acid mature protein (the signal sequence and pro-sequence are cleaved) - highly conserved during evolution, displays little sequence deviations in various species
What are the other members of the neurotrophin family?
-NFG, BDNF, NT-3, NT-4,NT-5, NT-6, NT-7
What led to the idea of more neurotrophins existing?
-NGF had no effect on some neurons, the retina brain etc. -that led to the idea that there are more neural growth factors -BDNF= brain derived neurotrophic factor purified from pig brains -held a promise in brain degeneration diseases therapies -then more neurotrophic factors identified, -humans have only NT3 and NT4 and NGF and BDNF, the otehrs are evolutionary dead ends
What is the neurotrophic factor hypothesis? (revisited after discovering that there were more than just one neurotrophin)
-The neurotrophic factor hypothesis: -Targets of innervation secrete limiting amounts of survival factors to generate a balance between the size of the target organ and the number of innervating neurons. -The neurotrophic factor hypothesis revisited: NGF: • is secreted by the target in limited quantities • binds to a ‘receptor’ on the terminal • is competed for by nerve terminals • signal passes retrogradely up the axon to the cell body • suppresses the apoptotic pathway
What is the receptor that NGF binds to and how was it discovered?
-p75 (low affinity receptor) -thought to be the key receptor -it failed to mediate any effects -so there must be another receptor
What are the neurotrophins and the receptors that they bind to that operate in humans? (the 4 mainly)
- NGF, BDNF, NT-3, NT-4,
- the receptors are TrkA, TrkC, TrkB and p75
- NGF binds to TrkA
- NT-3 binds to TrkC
- BDNF and NT-4 bind to TrkB
- all of them bind to p75
What is the interaction between the different receptors?
-so bind to a Trk receptor and p75 and bidn with different kinetics if both recptors are expressed, so can exert different effects depending on which receptors are expressed on a cell
How does the signal from the DRG get to the cell body work? (describe the experiment)
- put DRG neurons in the middle of the chamber, then neurons grow through grease, the axon tips were in an isolated compartment compared to cell body
- can put things into the cell body bit that wouldn’t on the tips and vice versa
- the only NGF neurons saw was at the tips= neuronal survival is mediated by feeding teh distal part of the axon, so proved that the signal si gnerated at the tip and maintains surivival
- NGF supports axon growth of even when other parts of the neuron are starved of NGF
- NGF is competed for by nerve terminals
- NGF can act locally to regulate growth events
- NGF sends a “retrograde signal” back to the cell body to support survival
- The molecular nature of this retrograde signal was not resolved for many years
What is the theory about Trk receptor signaling and retrograde transport? (the ones that are wrong)
1.The ‘domino’ model, a wave of ligand-independent Trk phosphorylation that is retrogradely propagated 2.The ‘retrograde effector’ model Local signals from Trk receptors are propagated to the cell body
What is the theory about Trk receptor signaling and retrograde transport? (the one that is right)
- The ‘signaling endosome’ model
- The ligand–receptor complex internalizes and forms specialized endosomes that serve as platforms for continued Trk signaling and are transported retrogradely to the cell body.
- internalized NGF and Trk are internalized and can act on the cell body
- signalling endosomes= the internalized Trk and NGF complex
What are the details of Trk receptor signaling and retrograde transport?
- Signaling at the cell body Trk-containing endosomes reach the cell body and activate signaling pathways which promote survival.
- Retrograde neurotrophin signalling: Signalling endosomes are platforms for unique signalling
- Local signaling at the axon Local activation of the PI3K and Erk1/2 pathways promote both axon outgrowth and receptor endocytosis.
What is thought to be involved in Huntington’s, Parkinson’s etc?
- Changes in retrograde transport along the axon can lead to defects in supply and clearance
- Neuronal function and survival depend on retrograde axonal transport
- A significant slowing of retrograde axonal transport is observed at early stages of disease in several neurodegenerative models, consistent with a pathogenic role.
- in huntingtton’s parkinson’s and alzheimers= there is a problem with the signal being transported back and the cell dies= unclear if primary reason or secondary effect
What is Tkf receptor signaling like?
- NGF signaling: -Multiple pathways activated: - axonal specific - cell body specific
- Gene expression activated when NGF present: Inhibition of apoptosis - intrinsic pathway
- Absence of NGF signaling pro-apoptotic pathways can be activated (intrinsic pathway)
- NGF- to Trk receptors = then pathways activated, these would converge and increase the expression of anti apoptotic genes, so impact the intrinsic pathway of apoptosis
- also blocks expression of pro apoptotic genes = so inhibits cell death overall! -in the absence of NGF = apoptosis triggered and more likely as pro apoptotic genes expressed!
How is neurotrophin signaling specific?
- The different neurotrophins support the growth and survival of different populations of neurons - The selective responsiveness allows the neurotrophins to regulate survival and synapse remodeling of different populations of neurons within the same region of target tissue. -different neurotrophins supported different neuronal populations= selective responsivness to neurtrophins -can have different neurotrophins in the same regions can have different effects =good
What population does NT-4 act on? (all connected to the DRG)
- hair follicle
- Each neurotrophin supports the survival of differing somatosensory receptors
- These specific effects have been determined from both tissue culture studies and the analysis of knockout mice.
What population does BDNF act on? (all connected to the DRG)
- Merkel disk -mechanosensation
- Each neurotrophin supports the survival of differing somatosensory receptors
- These specific effects have been determined from both tissue culture studies and the analysis of knockout mice.
What population does NGF act on? (all connected to the DRG)
- free nerve endings, pain signalling
- Each neurotrophin supports the survival of differing somatosensory receptors
- These specific effects have been determined from both tissue culture studies and the analysis of knockout mice.
What population does NT-3 act on? (all connected to the DRG)
- muscle spindle
- Each neurotrophin supports the survival of differing somatosensory receptors
- These specific effects have been determined from both tissue culture studies and the analysis of knockout mice.
What is the role of NGF? (final)
NGF: • is secreted by the target in limited quantities • binds to a ‘receptor’ on the terminal • is competed for by nerve terminals • signal passes retrogradely up the axon to the cell body • suppresses the apoptotic pathway
What are other trophic families?
-Many are trophic factors for neuronal and non-neuronal cells -Each have their own receptors & signalling pathways -Each affect specific subsets of neurons
Summary:
-Neurotrophins: -Identified through their survival promoting activity -Now rarely studied in that context -Take on distinct roles in the mature nervous system
