Final: Chapter 7 Flashcards
____ is a family of molecules that promote neuron survival
neurotrophins
Survival of neurons helped by
interaction with target tissue
3 ways for cells to die
1) Apoptosis
2) Autophagy
3) Necrosis
Apoptosis
PYKNOSIS: chromatin in crescent shape at nuclear membrane
proteins cross linked, apoptotic bodies pinch off, and are eaten by macrophages
*Surrounding cells protected
Experiment: TUNEL (UTP nick end labeling)
enzyme attaches labeled nucleotides to exposed DNA ends `
Autophagy
cytoplasm fills with autophages/lysosomes before pyknosis
Later stages of programed cell death
Necrosis
mitochondria stop making energy
swelling and explosion of soma
*Effects neighboring cells
Experiment: TUNEL and BrdU
ISEL labeling
BrdU: label synthesis of mitosis
TUNEL: counts pyknotic cells
Most apoptotic cells in ventricular zone where birth is happening
Neural crest cluster on NON-ADHESIVE surface =
increase in TUNEL labeling
Survival of neurons depends on amount of
target tissue
Experiment: Chicken DRG
Less cell death if limb
Remove limb: Few DRG/MN (increased death)
add limb: More DRG/MN (decreased death)
Significance? Target provides survival factor
Experiment: Reduce neurons that go to target rather than target itself. Does death happen?
No, not pre-oriented to die, need to reach target
Experiment: Hamburger’s mouse tumor
Part 2: Is it soluble?
Sarcoma
put inside a chicken.
Increase survival of DRG sensory neurons and sympathetic ganglion cells (SGC).
Part 2: Tumor of choriallontoic membrane
Tumor not in contact with sym/sensory ganglia, BUT share the same blood supply, strong growth effect
Therefore, it’s soluble!
Experiment: The snake venom
Snake venom breaks down nucleic acids, use to see fi proton or NA have growth factor
1) DRG and sarcoma + Snake venom
2) DRG + snake venom
Results?: growth in both! But loving the snake venom. Snake venom is a growth factor.
Where? NGF in salivary glands.
Why is pro-NGF cleavage a big deal?
cleaved= bioactive version.
BDNF = \_\_\_ ganglia NFG= \_\_\_ ganglia
BDNF Vestibular
NGF cochlear
Remove TrkA
no response to NGF
Transactivation
TRK receptors activated in absence of neurotrophins
NGF in PCl2 cells
protein phosphorylation
onocogene 3T3
genetic rearrangement, fuses with tyrosine kinases (TRKA), induces kinase activity and phosphorylates stuff
RTK: receptor dimer, neurotrophins bind with specificity
PCL2 cells with TrkA -/- KO
no TrkA? cell death, fewer axonal projections, unresponsive to NGF
Actinomycin-D
blocks TRANSCRIPTION by binding to DNA preventing the movement of RNA polymerase
Cycloheximide
prevents TRANSLATION by blocking peptidyl transferase reaction on ribosomes
______ and ____ rescue neurons after NFG deprivation
Actinomycin-D and Cycloheximide
Why can Actin-D and Cyclo-H prevent cell death?
because cell death needs proteins
When NFG removed, sympathetic neurons…
DIE
Experiment: anti-NGF beads
1) Anti-NGF = 22% survive
2) Anti-NGF, but put in some NGF = 95% survive
3) Use the beads= 85% survive
Why the beads? make stationary to see if retrograde transmit needed.
Experiment: block antibody in somata of sympathetic neurons,
only 60% neurons survive
Trk internalization
1) NGF binds to TrkA, dimerization and autophosphorylation of receptor
2) Ligand-R has clathrin dependent endocytosis. Effector proteins recruited and endo-vesicles activated.
3) Endosome is transported retrogradely along microtubules by dynein motor.
4) endosome at cell body, signal to control transcription
Experimental evidence (x3) for Trk receptor internalization and such
1) NGF/Trk A/ effector colocalized within endosomal…?
2) increase death when dynein motor protein interfered with
3) block TrkA phosphorylation = decrease survival
___ initiates cell death with coreceptor ____
p75 NTR
Sortilin
___ binds with high affinity to sortilin and p75 NTR
proneurotrophins
What happens if you block proNGF and sortilin
decrease in cell death
The proNGF/sortilin/p75NTR survival pathway
TRAF6
NIK, stuff with K in it
Activates anti-apoptotic genes
The proNGF/sortilin/p75NTR death pathway
NRIF: grabs TRAF6
Ask1 –> JNKK (J’s activated)
Jun- phosphorylated
Pro-apoptotic genes activated
Name the 3 big survival pathways
AKT
RSK
ERK (MAPK)
Name the 1 apoptosis pathway
JNK
Experiment:
1) Remove NGF
2) +JNK
3) +ERK
4) + p21
1) death
2) death
3) life
4) Life, p21 is a cyclone dependent kinase inhibitor (inhibits cdk, which is part of death pathway)
What keeps everything in mitochondria?
Bcl-2
What inhibits JNK?
MAPK
C. Elegans Death pathway
Proapoptotic EGL-1 inhibits Ced-9
Ced-9 is life: inhibits Ced 4
Ced 4: activates Ced 3
Ced 3: protease, cuts after asparatate residue to activate apoptosis
What inhibits Bcl-2 to cause death in mammals?
Bax
What inhibits IAP so it stops inhibiting capase-9?
Smac
mammal pathway:
Bax —-| Bcl-2 –| cyto c —> Apaf 1 –> caspase 9 –> Caspase 3 –> Apoptosis
What keeps apaf-1 and bax locked up?
Bcl-X
What 3 things does Bcl-2 have in mitochondria
cyto c
SMAC
AIF
What does BAD do once dephosphorylated?
bind to Bcl-x
frees Apaf-1 and Bax
What does Apaf-1 do once it’s free
forms apoptosome with cyto c
What does bax do once it’s free?
Free everything from Bcl-2
Smac:
binds IAP, free procaspases 9
What cleaves procasapase 9?
apoptosome (apaf-1 and cyto c)
Caspase 9
cleaves procaspase 3 into active caspase 3
Caspase 3
targets ICAD to release CAD
Cad joins with __ and goes to the nucleus to do what?
AIF
cleave DNA
How does lack of NGF cause cell death?
cycline D cdk4 pathway
In life pathway: what keeps BAD phosphorylated and inactive
NT kinase activity
LIFE: NGF/TrkA: activates SOS and RAS, which decreases
c-Jun
LIFe: p21
cdk inhibitor
life: p75
no sortiln, TRAF6, NFB
Life: ERK, RSK, and AKT promote what
Bcl2 keeping stuff in mitochondria
KO bcl-x and bax vs. KO only one
a lot of cell death vs. a LOT of cell death
Experiment: NMJ treated with Curare
more cells live
Less activity in MOTOR = more survival
Why? different systems, different rules
Experiment: cochlea removal in chickens
Sensory relies on electrical activity, but the death doesn’t happen until E2 when you would actually use it.
Experiment: Cochlea removal in a gerbil
p7- 50% neuron loss
p9- no neuron loss
Activity for survival, once activity already happen, not need to die.
Experiment: induce depolarization by adding KCl to neurons
BDNF goes up, more neuron survival
Experiment: add BDNF antibody
trophic influence of depolarization eliminated
Estrogen vs. testosterone
estrogen: pro-apoptotic, kill male genitals
Testosterone: survival factor
Experiment: Female and testosterone
SNB and MN death decreases
Experiment: male castrated and flutamide (androgen antagonist)
SNB and MN cell death increases
Depolarization
survival via NT release
activity (for sensory anyway) increases survival
