Cell Death

Question 1

is there a relationship btwn targets of innervation and the pool of innervating neurons?

Answer 1

remove limb bulb (target) from one side of chick embryo
-results: many fewer neurons in the region of the spinal cord that would originally internervate the ‘missing’ limb

Question 2

limb ablation and limb addition

Answer 2

-ablation resulted in smaller ganglia with removal of target
-addition (supernumerary) resulted in a larger ganglia

Question 3

what did Hamburger postulate about muscle and skin

Answer 3

muscle and skin made 2 different, specific agents/stimuli that control the development of their respective nerve centers

Question 4

how do ‘agents’ from each target reach their respective nerve centers? (recruitment model)

Answer 4

agents travel retrogradely to their respective nerve centers (brachial lateral motor columns and spinal ganglia)
-regulate the development of corresponding centers

Question 5

how do the agents regulate the development of these centers? (recruitment model)

Answer 5

-nerve centers send axons to their respective target areas
-axons would explore the size of the target area and send ‘agent’ to cell bodies
-promote the differentiation of adjacent undifferentiated cells, would then send more axons to target
-‘recuritment’ of undifferentiated cells would proceed until target is saturated with nerves

Question 6

recruitment model

Answer 6

targets of innernation ‘recruit’ naive cells in the spinal cord/spinal ganglia adjacent to the neuronal cell bodies to become neurons
-‘recruitment’ factors acquired by axons and determine cell fate of naive cells

Question 7

reduced and extra target material

Answer 7

remove limb bud: fewer neurons
additional limb bud: more neurons

Question 8

Hamburger’s model

Answer 8

1. decline in neuronal numbers is because many neurons that are initially present degenerate following ablation->atropy causes reduced numbers, not recruitment
2. cell death occurs not only after limb ablation, but as an ordinary and normal feature of embryonic development

Question 9

what is the molecular mechanism by which targets can regulate neuronal cell death?

Answer 9

neuronal survival requires acquisition of a target derived ‘survival factor’ which is present in limiting quantities, leading to competition for the survival factors by the pool of internervating neurons
-axons need to reach threshold of survival factor to survive, or they will die

Question 10

what are the predictions of Hamburger’s model

Answer 10

1. number of neurons arriving at single target increases, the number of survivors should stay constant, % of survivors should decrease
2. # of neurons arriving at single target decrease, # of survivors should stay constant, % of survivors should increase

Question 11

Mouse sarcoma 180

Answer 11

as it grows it gets invaded by nerves
-DRG/sympathetic ganglia closest to tumor get very large
-both sensory and sympathetic neurons invade tumor, but not other neurons (motor neurons)

Question 12

is the survival factor a diffusable/ soluble substance?

Answer 12

Exp: put tumor on other side of allantoic membrane instead of inside embryo (no direct contact w/ embryo)
-survival factor diffuses everywhere
-results: ganglia was still enlarged when placed on other side of membrane
-conclusion:survival factor is likely a diffusable factor

Question 13

fractionation procedures

Answer 13

-starting material is run through resin to fractionate
-fractons are assayed to identify desired molecules
-fractionate with diff resin->assay again (repeat)

Question 14

what was the result of adding the survival factor to a chick dorsal root ganglia (halo assay)?

Answer 14

-when survival factor was added-> lots of extended neurites (halo)
-when no survival factor was added->dead ganglia

Question 15

phosphodiesterase experiement: is the survival activity a protein or nucleic acid?

Answer 15

-if it was a protein there should have been a halo
-if it was a nucleic acid, there should be no halo
-results: there was a HUGE halo

Question 16

what happened when a negative control (buffer) was added with phosphodiesterase (PDE)?

Answer 16

there was a huge halo
-this wasn’t expected because the control shouldn’t have any survival factor
-made researchers ask what the starting material that PDE was purified from->snake venom

Question 17

what is a better homolog for snake venom producing organs?

Answer 17

male mose salivary glands

Question 18

if we add extra NGF, neuronal cell death should decline and neuronal survival should increase (prediction #1)

Answer 18

Addition of NGF
-increased neuronal survival
-chemotaxis-growth of axons to site of injection

NGF is sufficient to promote neuronal survival

Question 19

if we remove endogenous NGF, neuronal cell death should increase and neuronal survival should decrease (prediction#2)

Answer 19

immunosympathectomy: remove sympathetic nerves
-resulted in first ever use of function blocking anitbodies
-is there a halo with the function blocking Ab-> NO

FB Ab resulted in complete destruction of sympathetic NS, partial destruction of sensory SN and no effect on motor neurons

Question 20

NGF must by synthesized in targets in proportion to the levels of innervation (prediction#3)

Answer 20

increased NGF levels and density of innervation supports the idea that production of NGF by target organs determines density of innervation

Question 21

NGF must be synthesized in targets at the right time in development (prediction #4)

Answer 21

-NGF mRNA and protein synthesis both start around the time that the axon arrives to cell body
-heart still synthesizes NGF, despite no growth cones arriving->NGF isn’t induced by arrival of growth cones

Question 22

how did researchers make larger quantities of NGF?

Answer 22

1.isolation of NGF cDNA as a probe to detect NGF mRNA on RNA blots
2.improved immunological assays to detect NGF proteins using monoclonal antibodies

Question 23

what target derived trophic factors act in the CNS?

Answer 23

brain derived neurotrophic factors
-65% amino acid homology to NGF
-both are neurotrophins

Question 24

are there more members of the neurotrophin family?

Answer 24

sequenced highly conserved regions shared btwn NGF and BDNF
-found that NT3 and NT4 were homologous

Question 25

PC12 cells

Answer 25

transformed cell line that differentiates in response to NGF but doesn’t require NGF for viability
-neural precursor to neuronal phenotype

Question 26

Is tyrosine phosphorylation involved with NGF?

Answer 26

tyrosine phosphorylation
-rapid increase in tyrosine phosphorylation correlates with NGF treatment
-resulted in 2 proteins on immunoblot

Question 27

what proteins do NGF interact with on the plasma membrane?

Answer 27

found2 proteins (158 kDa and 98 kDa)
-proposed that binded to a 85 kDa and a 60 kDa molecule
-would require 2 cross-linking events to occur (very rare)
-not a super plausible model

Question 28

Purify 158 kDa crosslinked complex to determine what was present

Answer 28

-add non radioactive NGF and covalent crosslinker (that was reversible)
-label all NGF containing complexes with radioactivity
-then reverse cross links
-if 85+60 model is right, we should see 85 and 60 bands
-results: no 60 kDa band existed and a 135 kDa protein was present

Question 29

what was the 135 kDa band

Answer 29

TrkA
-an orphan receptor
-NGF causes rapid tyrosine phosphorylation of trkA
-trkA rescues mutant PC12 cells that are unable to respond to NGF, allows good neurite outgrowth->shows that it is an important receptor of NGF

Question 30

where can the NGF signal be aquired

Answer 30

at the tips of the growing neuronal process to promote neuronal cell survival
-when there was no NGF at tips, there was very little neuronal survival
-when there was NGF at tips, there was significant neuronal survival

Question 31

how is the survival signal relayed back to the cell body?

Answer 31

through signal cascades carried through the axon (kinesin)

Question 32

neuronal cell death can be delayed by blocking protein synthesis

Answer 32

-NGF represses cell death program, it is a growth factor

Question 33

universal mechanism of animal development

Answer 33

C. elegans
-genome is fully sequenced
-131 cells always die

Question 34

ced-3 and ced-4

Answer 34

mutant: animal cells with no cell death, have too many cells
normal function: promote cell death

Question 35

ced-9

Answer 35

mutant: animal cells with too much cell death
normal function: prevent cell death

Question 36

ICE

Answer 36

interleukin converting enzyme; part of the immune system
-related to ced-3
-cut in precise location to activate IL-1 beta

Question 37

how do ced-3/ICE medicate cell death in fibroblasts

Answer 37

mouse ICE cDNA: a lot of cell death
worm ced-3 DNA: a lot of cell death
-suggests that ced-3/ICE mediates cell death via protease function
-cell death machinery is in place, just needs a trigger

Question 38

how do ced-3/ICE mediate cell death in neurons

Answer 38

no NGF (survival) and crmA (ICE inhibitor)=no cell death

Question 39

what protein is ced-9 similar to in mammals?

Answer 39

bcl-2
-both are anti-cell death cells (survival cells)
-ced-9 worms transfected in WT mouse bcl-2 cDNA = normal cells of cell death
-bcl-2 can promote cell survival in worms and mamilian neurons

Question 40

apoptosis in mouse

Answer 40

1. influx of Ca2+ from outside of cell and release from Ca2+ from internal stores, raising intracellular Ca2+ levels
2. high Ca2+ triggers release of diablo protein from mitochondria
3. diablo binds to IAP (inhibitors of apoptosis proteins), can no longer block caspases
4. caspases signal and apoptosis results

Question 41

how does bcl-2 inhibit apoptosis

Answer 41

blocks release of diablo
-cells need lots of bcl-2:bcl-2 heterodimers to stop apoptosis
-if there is little bcl-2:bcl-2 heterodimers, and there is more bax the cell death pathway will be active

Question 42

does NGF signalling really induce bcl-2 transcription?

Ricco et al., Science 1999 (section paper)

Answer 42

-CRED is a transcription factor that is necessary for NGF signalling
-CRED promotes survival, even in absense of NGF (acts downstream of NGF-trkA)
-NGF mediated induction of bcl-2 gene transcription requires CRED
-Conclusion: NGF signalling activates CRED, which induces bcl2 gene transcription, resulting in cell survival

Question 43

cortical (CNS) interneurons

Answer 43

if transplant various numbers of cells into animals, ~40% of cells die regardless of how many are transplanted (doesn’t fit target derived neurons)
-cortical interneuron death is determined intrinsically