Neurotrophins

Question 1

neutrophil hypothesis

Answer 1

neurone survival depends on a supply of neurotrophic factors synthesised in limiting amounts in their target fields

Question 2

stages in neurodevelopment

Answer 2

1. determination
2. proliferation
3. migration
4. axon elongation
5. synapse formation
6. synapse rearrangement

Question 3

trophic interactions support which stages?

Answer 3

survival of subset of neurones

formation + maintenance of appropriate connections

growth of axonal + dendritic branches to support the connections

Question 4

neurotrophins

• features
• best studied example

Answer 4

family of trophic factors for neuronal survival

secreted by target cells
(neurones or innervated tissues)

e.g. Nerve Growth Factor

Question 5

nerve growth factor (NGF)

- discovery

Answer 5

in a limb bud experiment:
implanted some tumour cells as a control
-> one type of tumour caused growth of nerves toward tumour
.:. tumour secreted an attracting factor

used snake venom (phosphodiesterase source) to destroy any Ras
-> massive neurone outgrowth
.:. snake venom = rich in NGF sources

-> then purified NGF

Question 6

NGF in vivo

- effects on cervical ganglia

Answer 6

if treated daily with NGF anti-serum
-> atrophy + unhealthy
+ loss of neurones

control
-> sustained connections + survival of neurones

Question 7

NGF in vivo

- neurone population size

Answer 7

inject extra NGF into immature animals
-> enlargement of sympathetic ganglia

NGF can determine size of a neuronal population

Question 8

action of NGF is concentration-dependent

Answer 8

limiting conc of NGF
-> neurones compete for it
-> only a proportion survive
= rest die

only enough NGF produced to promote survival of required no. of neurones

Question 9

NGF exerts a local action on neurites

Answer 9

neurites grow if NGF source present

neurites regress if NGF then removed

contact with neuronal cell body NOT required

Question 10

NGF structure

Answer 10

polypeptide ~118 aa
dimer

secreted as a larger precursor
-> processed extracellularly to give NGF

each NGF monomer
= 4 anti-parallel beta strands connected by 4 loops

b strands connected by 3 cysteine disulphide bonds
= a ‘cysteine knot’

Question 11

NGF and TrkA

Answer 11

NGF binds to d5 immunoglobulin domains on TrkA extracellular domain

-> activates intracellular Tk domain

Question 12

NGF and dimeric TrkA

Answer 12

binding dimeric NGF stablises dimeric TrkA

• > induces structural changes
• > activates TK by autophosphorylation

Question 13

crystal structure of NGF in complex with d5 on TrkA

Answer 13

NGF core binds to C terminus on d5

NGF N terminus binds to ABED sheets of d5

Question 14

activated TrkA

- promotes signalling pathways

Answer 14

PI3 kinases
-> cell survival

Ras/Raf pathway
-> neurone outgrowth + neuronal differentiation

PLC
-> activity-dependent plasticity

Question 15

how does TrkA interact with multiple targets?

Answer 15

(after autophosphorylation)
via phosphotyrosine
->

Question 16

activity-dependent plasticity

Answer 16

synapses strengthened or remade depending on amount of nerve activity

Question 17

ras-raf pathway

- up to activating Ras

Answer 17

1. phosphoTyr residues on TrkA bound by SH2 domains in SHC protein
2. SHC is Tyr phosphorylated
   - > bound by SH2 domains in ‘adaptor protein’ Grb2
3. Grb2 binds Sos protein which links to membrane-bound Ras
4. binding promotes exchange of GTP for GDP
   = activates Ras

Question 18

ras-raf pathway

- post active Ras

Answer 18

1. active ras recruits cytoplasmic Raf kinase
2. activates bound Raf
   - > phopshorylates serine residues on MEK
3. MEK phosphorylates ERK
4. phosphoERK phosphorylates multiple cytoplasmic targets OR migrates to nucleus to influence TFs

Question 19

retrograde transport of NGF-TrkA signalling

Answer 19

NGF-TrkA complex taken up by neurones
-> trafficked to cell body

internalisation via endosome formation

Question 20

p75NTF

- features

Answer 20

2nd receptor of NGF

balances survival-vs-death decisions

low affinity for processed NGF
- higher affinity for unprocessed form

associated with neurone apoptosis

Question 21

p75NTF

- mechanism

Answer 21

couples to multiple signalling pathways
- different to TrkA signalling cascades:

> RhoA
-> growth cone collapse (via apoptosis)

> c-Jun
-> cell death (apoptosis)

> NF-kB
-> cell survival

Question 22

outcome of NGF signalling depends on..

Answer 22

NGF concentration

which receptors are present

intracellular signalling molecules expressed + in vicinity of receptors

longer term changes from internalised + trafficked NGF-TrkA

Question 23

NGF doesn’t support all neurones

Answer 23

doesn’t have widespread effects in CNS

does support population of cholinergic neurones in basal forebrain

Question 24

brain-derived neurotrophic factor (BDNF)

Answer 24

structurally homologous to NGF (50% sequence identity)

released as a precursor proBDNF peptide

• > processed into BDNF
• > binds to TrkB

(proBDNF binds to p75 receptor)

Question 25

neurotrophins

• and Trk receptors
• and p75 receptor

Answer 25

Try receptors are homologous
- distinct but overlapping specificities

TrkA - sensory + sympathetic neurones
TrkB and TrkC - more widely distributed

all neurotrophins share same p75 receptor

Question 26

which neurotrophins bind to which:
TrkA?
TrkB?
TrkC?

Answer 26

NGF

BDNF
NT-4/5
NT3 (weakly)

NT3

Question 27

neurotrophins collaborating with other players in neuronal development and maintenance
- example

Answer 27

Slit1 + BDNF + Notch

- encourage branching of dendrites in brain

Question 28

Glial-derived neurotrophic factor (GDNF)

- features

Answer 28

40kDa dimer

preferentially binds to GFR-alpha1

crucial role in development + survival of various neurone populations
- notably dopamine neurones

Question 29

GDNF

- Parkinson’s disease

Answer 29

therapeutic candidate

involved in survival of dopamine neurones
- die in Parkinson’s disease