19 - Keeping Neurons Alive: Neurotrophin Signaling Flashcards
What discovery led to Rita Levi-Montalcini receiving the Nobel Prize?
The discovery of NGF
What type of molecule is a trophic factor?
A small protein or proteins
True or false: trophic factors are secreted by a few select cells
False: they are secreted by multiple cell types
How are trophic factors secreted?
As an inactive precursor
How are the precursors of trophic factors activated?
Through cleavage or fragmentation
True or false: precursors of trophic factors are inactive
False: they also have functions (although they are different that the functional trophic factor)
How are trophic factors transported?
Through the blood and lymph systems
What do growth factors do?
Stimulate cells to divide or increase in size
What is hyperplasia?
Stimulating cells to divide
What is hypertrophy?
Simulating cells to increase in size
What are the effects of trophic factors?
Cell differentiation, survival, expression of a particular cellular phenotype, and cellular morphological plasticity
True or false: a growth factor can act as a trophic factor
True: some can act as both
True or false: a trophic factor can act as a growth factor
True: some can act as both
True or false: growth factors can have the same functions in vivo vs. in vitro
False: they can have different functions
What are the functions of growth factors in vitro?
Proliferation, differentiation, chemo-attraction, cell death, and cell migration
What are the functions of growth factors in vivo?
Early development, tissue differentiation, wound healing and tissue repair, immune responses, and mediating sex/other hormones
How can differences arise between the functions of the same growth factor?
Based on the study (cell culture vs intact organism) and scale
What are neurotrophins?
A family of proteins essential for the development of the vertebrate nervous system
What are some examples of neurotrophins?
NGF, BDNF, NT3
What does NGF stand for?
Nerve growth factor
Which cells produce neurotrophins?
All cells of the nervous system (neurons, glial cells), ependymal cells, blood vessel endothelial cells, and cells from innervated tissues (muscle, epidermis, etc.)
What are some examples of glial cells?
Astrocytes and oligodendrocytes
True or false: neurotrophic factors come from a specific region in the body
False: they come from all over the body
When was Victor Hamburger’s work done?
1930s
What experiments did Victor Hamburger do?
He added or removed limb buds to see how this affected neuron growth and development
What were the results of Hamburger’s experiments?
Less neurons grew when the limb buds were removed, and more neurons grew when an extra limb bud was added
What was the conclusion of Hamburger’s experiments?
The peripheral tissue was sending signals for the neurons to grow and divide
How did Rita’s hypothesis differ from Hamburger’s hypothesis?
Rita believed that there was a degenerative process, while Hamburger believed there was a failure of differentiation
What evidence did Rita find for her hypothesis in the 1940s?
Neurons regularly underwent apoptosis during development
According to Rita’s hypothesis, what happened when a limb bud was removed from the embryo?
There was increased cell death
According to Hamburger’s hypothesis, what happened when a limb bud was removed from the embryo?
There was decreased cell growth / proliferation
What was Rita’s hypothesis regarding nerve development?
Feedback signals required for neuronal survival are in limited supply
In development, which neurons will survive?
The ones that take up enough neurotrophic factor
What is meant by a “matching” of target area?
The target cells will produce enough neurotrophic factors to match the number of target cells
True or false: neurotrophic factors are only important in development
False: they are also important after development
How are neurotrophic factors used after development?
Axonal processes can compete for neurotrophic factors
What determines whether a neuron will take up significant neurotrophic factor?
How active the synapse is
What alters how active a synapse is (for taking up neurotrophic factors)?
Experience and stimulation
What happens to neuronal development when the chick embryo was paralyzed?
Only some of the neurons survived
What was the conclusion of the experiments involving paralyzing the chick embryo?
The neuron and muscle together determine how the muscle signals to the neuron (regarding neurotrophic factors)
What is an important regulator of neurotrophic factor release?
Muscle activity
What experiments did Rita perform in the 1950s?
Transplanted tumors induced potent growth of the nervous system in chick embryos
What was the conclusion of Rita’s experiments in the 1950s?
The tumor released a nerve growth-promoting factor that acted on the nerves (trophic growth factor hypothesis)
What is the significance of tumors not needing direct contact in Rita’s experiments (in the 1950s)?
There must have been a secreted factor (trophic growth factor hypothesis)
What was the advantage of using sarcoma tissue?
There was more material to work with
Who was Stanley Cohen?
A biochemist hired to help purify NGF
Why was Stanley Cohen hired to the Hamburger lab?
They needed a biochemist to help purify NGF
What was in the compounds of the extract that Cohen purified?
Proteins and nucleic acids
What did Cohen do to determine if NGF was the protein or nucleic acid?
Added snake venom to inactivate the nucleic acids
Why was snake venom used to determine if NGF was a protein or nucleic acid?
Snake venom has nucleic-acid degrading enzymes
What was the problem with using snake venom?
It contained more nerve growth-promoting activity than the tumor itself
Besides snake venom, what is a rich source of NGF?
Mouse salivary glands
True or false: NGF is a potent molecule
True: it can lead to rapid changes with a small concentration
What was needed to study and purify NGF?
A rapid assay (faster than adding sarcoma pieces to embryo)
How come a rapid assay was needed to study NGF?
Extracting proteins can degrade quickly and cannot be measured
What was the rapid assay used to identify NGF?
Root dorsal ganglion cells from chicken embryos
Besides NGF, what did Cohen find in the NGF extracts?
EGF
What does EGF stand for?
Epidermal growth factor
What was the effect of EGF on mice?
Teeth erupted, and eyelids opened sooner than normal
True or false: EGF can only affect epidermal cells
False: it could also affect other types of cells
What was the conclusion regarding EGF acting on multiple types of cells?
Any cell that has an EGF receptor can respond to EGF
What did Cohen discover about the EGF receptor?
EGF and the EGF receptor, when bound together, entered the cell
What is the structure of NGF?
Dimeric, with binding along a hydrophobic interface
How did Rita and Cohen prove that NGF was the intended factor?
By using anti-NGF antibodies to neutralize it
How could NGF be verified using modern technology?
By using a knockout mice
What happened when NGF was blocked by an antibody?
There was no peripheral ganglia
What are PC12 cells?
A neuron-like cell line from a rat tumor
Without NGF, how do PC12 cells grow?
Similar to tumors
With NGF, how do PC12 cells grow?
Similar to neurons
What was the conclusion regarding adding NGF to PC12 cells?
NGF stimulates neuronal phenotypes separate from the prevention of cell death
According to the PC12 model, what neural phenotypes can be gained upon addition of NGF?
Neurites, sodium action potential, stop cell division, synthesize neurotransmitters, and create neuronal specific proteins
Why is it difficult to study NGF in vivo?
Neurons die when NGF is knocked out
What was done to get around the difficulty of studying NGF in vivo?
BAX was knocked out to inhibit cell death
What is BAX?
A gene involved in mitochondrial death and apoptosis
What happened when both NGF and BAX were knocked out?
The cell survived, but could not differentiate into a neuron (based on the NGF functions)
What happened when NGF was knocked out?
The cell died (no survival)
True or false: NGF has branched effects
True: it can lead to multiple phenotypes
What are the branched effects of NGF?
Control neuronal phenotype, and prevent cell apoptosis
What was done to study the signaling pathways of NGF?
Study the known signaling pathways in PC12 cells
In PC12 cells, what was added that mimic the addition of NGF?
Addition of constitutively active Ras protein
What happens when anti-ras was added to PC12 cells?
They did not get the neuronal phenotype
What was the control in experiments with anti-ras and PC12 cells?
Another antibody (anti-something else)
What was the purpose of the control in the experiments with anti-ras and PC12 cells?
Demonstrate that adding the antibody would not change the system (had to be anti-ras)
What was the conclusion of the studies with anti-ras in PC12 cells?
NGF uses a proto-oncoprotein pathway for neuronal survival and development
What is TrkA?
The receptor tyrosine kinase for NGF
What receptor binds to NGF?
TrkA
What happens to TrkA when NGF binds to it?
It dimerizes and leads to autophosphorylation of the RTK
What pathways are activated by TrkA?
The PI3K, ras, and PLC pathway
What does the PI3K pathway lead to?
The activation of Akt
What does the Ras pathway lead to?
The activation of the MAP kinases
What does the PLC pathway lead to?
The activation of intracellular calcium and PKC
True or false: TrkA is a proto-oncogene
True: it can lead to constant activation of the Ras pathway if mutated
What is the structure of the TrkA oncoprotein?
It is fused with tropomyosin, which keeps it constitutively dimerized and active
What happened when the constitutively active TrkA receptor was put into non-neuronal cells?
It led to uncontrolled mitogenesis
What happened when the constitutively active TrkA receptor was put into neuronal cells?
It mimicked NGF action
How are the different Trk isoforms formed?
Through alternative splicing
What is the significance of alternative splicing in Trk receptors?
It leads to different isoforms, which alters the downstream signaling pathways
True or false: RTKs have similar extracellular domains
False: the extracellular domains are fairly diverse
True or false: RTKs have similar intracellular domains
True: the intracellular domains are fairly similar
What is the preferred ligand for TrkA?
NGF
What is the preferred ligand for TrkB?
BDNF and NT-4
What is the preferred ligand for TrkC?
NT-3
What is a preferred ligand?
A ligand that binds with high affinity to the receptor
What is a non-preferred ligand?
A ligand that binds with low affinity to the receptor
What is a non-ligand?
A ligand that does not bind to the receptor
Besides Trk receptors, what receptors can NGF bind to?
p75NTR
What does p75NTR stand for?
p75 neurotrophic receptor
How does p75NTR interact with Trk receptors?
p75NTR can interact positively or negatively with Trk
True or false: Trk can only transmit positive signals
True: it causes the cell the survive
True or false: p75NTR can only transmit positive signals
False: it can also send negative signals, such as promoting apoptosis
True or false: Trk can only bind to mature NGF
True: NGF needs to be cleaved to bind to Trk
True or false: p75NTR can only bind to mature NGF
False: it can also bind to pro-NGF
When pro-NGF is bound to p75NTR, what signal is transmitted?
A negative signal
When NGF is bound to p75NTR, what signal is transmitted?
A positive signal
Besides pro-NGF, what binds to p75NTR to promote a negative signal?
Sortilin
What does sortilin do?
It promotes a negative pathway when bound to p75NTR
Which receptor does pro-NGF have a higher affinity for?
p75NTR
Which receptor does NGF have a higher affinity for?
TrkA
True or false: p75NTR can bind to all classes of neurotrophins
True: it can bind to all neurotrophins in a mature or pro form
True or false: p75NTR only has preferred ligands
True: it has no non-preferred ligands or non-ligands
In terms of neurotrophic receptors, what is important for determining the number of surviving cells?
The ratio of Trk and p75 receptors
True or false: p75 plus Trk can lead to greater cell survival than just Trk
True: this combination of effects leads to an overall greater effect
How is the ratio of Trk to p75 seen in muscle contraction?
Motor neuron axons can retract with pro-BDNF, and extend with BDNF (through their receptors)
What is the importance of motor neuron axons retracting and extending?
It stabilizes or destabilizes the synapse at the neuromuscular junction
True or false: BDNF is a candidate for a therapeutic agent in Parkinson’s
True: it has been shown to promote neuroprotection and neuroregeneration
What was found to be neuroprotective in Parkinson’s patients?
Physical training
What mediates physical training being protective in Parkinson’s disease?
BDNF and TrkB
How is p75 seen in HIV?
There is an increase in p75, leading to increased cell death, and impaired cognitive function
What is different about neurons (compared to other cell types) that leads to the different phenotypes observed?
Different combinations of signaling compartments (pathways) to act on
