Neuroscience lectures 6-7 - Synapse Formation

Question 1

Describe the development of the neuromuscular junction

Answer 1

1) Motorneuron growth cone approaches developing muscle
2) Nerve-muscle contact formed

3) Growth cone begins to differentiate into a presynaptic terminal
- accumulates neurotransmitter-containing vesicles
PRESYNAPTIC DIFFERENTIATION

4) Initially many axons may bind to one muscle fibre, but only one will stabilise, others will be eliminated
- Schwann cells start to myelinate axon

5) Post-synaptic muscle membrane invaginates to form post-junctional folds that accumulate neurotransmitter receptors
POSTSYNAPTIC DIFFERENTIATION

Question 2

What is secreted by muscle and schwann cells in early synaptogenesis?

Answer 2

• Synaptic basal lamina

- lies between nerve and muscle at forming synapse

Question 3

What is the role of the synaptic basal lamina?

Answer 3

It contains signals for both pre- and post- synaptic differentiation

Question 4

What is a good source for purifying molecules involved in synapse development?

Answer 4

Electrocytes found in the electric organ of electric fish such as eels
- Rich source of synaptic material

Question 5

Where is neural agrin secreted and what is its main function?

Answer 5

• Secreted by motorneurons during synaptic development

- Induces ACH receptor clustering

Question 6

How are different isoforms of agrin produced?

Answer 6

Alternative splicing

Question 7

Where are some other forms of agrin present?

Answer 7

In the muscle (mAgrin) and the CNS

Question 8

What splice site gives the most active form of neural agrin?

Answer 8

The B/z splice site

Question 9

What is the mechanism of Agrin action in terms of receptor binding?

Answer 9

2 parts of the nAgrin receptor:

1) Muscle-Specific Kinase (MuSK)
2) Low-density lipoprotein receptor-related protein 4 (Lrp4)

• MuSK expressed on developing myotubes
• Agrin binds to Lrp4, which causes myotubes to autophosphorylate MuSK
• Results in signalling that induces ACHR signalling

Question 10

What in the downstream signalling pathway of MuSK/Lrp4?

Answer 10

• MuSK autophosphorylation recruits Dok-7 adaptor protein that becomes tyrosine phosphorylated
• Dok-7 recruits Crk and Crk-L that interact with a yet unknown pathway resulting in ACH receptor phosphorylation
• ACHRs cluster

Question 11

What is the role of Rapsyn in MuSK-Lrp4 signalling?

Answer 11

• Peripheral membrane protein - attached by lipid anchor
• Clustered in response to nAgrin-MuSK/Lrp4 signalling
• Interacts directly with ACH receptors in 1:1 ratio
• Also interacts with cytoskeletal proteins, dystrophin, utrophin etc:
  > Regulates clustering of these proteins
  > Anchors clustered receptors to cytoskeleton

Question 12

What are the 3 ways that ACH receptors accumulate in the muscle membrane directly opposite a nerve terminal?

Answer 12

1) Redistribution of existing receptors (nAgrin/MuSK/Lrp4/Rapsyn)
2) Synthesis of new receptor proteins by subsynaptic nuclei
3) Switching off expression of extrasynaptic receptors

Question 13

What pathway regulates synthesis of new receptor proteins?

Answer 13

Neuregulin-ErbB

Question 14

What type of molecule is Neuregulin-1 and where is it found?

Answer 14

Member of the epidermal growth factor family

- secreted by motorneuron into the synaptic basal lamina

Question 15

What type of ErbB receptor binds neuregulin-1 and where is it found?

Answer 15

A ErbB2 tyrosine kinase receptor

- on muscle membrane

Question 16

What does Neuregulin-1/ErbB2 signalling cause?

Answer 16

• Induces transcription of ACH receptor subunits in subsynaptic nuclei by regulating specific transcription factors

Question 17

What binding is also required for neuregulin-1/ErbB2 signalling?

Answer 17

nAgrin/MuSK/Lrp4

Question 18

How are ACH receptors in extrasynaptic nuclei repressed during early electrical activity?

Answer 18

• ACH binds to ACHR
• Depolarisation
• Ca2+ channels open
• Ca2+ entry
• Activation of Ca2+ dependant kinase pathway resultins in silencing on myogenin
• This is a transcription factors that regulates ACH receptor subunit gene expression

Question 19

What signalling is required for pre-synaptic differentiation?

Answer 19

nAgrin/MuSK/Lrp4 signalling

Question 20

What does this suggest about the signalling pathway that induces pre-synpatic differentiation?

Answer 20

nAgrin activation of MuSK must trigger a retrograde signalling pathway from muscle back to nerve

Question 21

What are some candidate retrograde signalling molecules?

Answer 21

• Laminin-beta2
• Collagen IV
• Fibroblast growth factors (FGFs)

Question 22

What experimental evidence suggest there are other molecules involved?

Answer 22

Loss of any of the above molecules only has mild effects

Question 23

What disorders are related to the neuromuscular junction? What are the common symptoms?

Answer 23

• Broad range
  > Myasthenic disorders e.g myasthenia gravis
  > Neuromuscular junction protein disorders
• Weakness and fatigability of skeletal muscle

Question 24

What are neurmuscular disorders usually caused by?

Answer 24

• Gene mutations

- Autoimmune attack of proteins (pre- or post synaptic, basal lamina)

Question 25

Dysfunctions in which proteins make up the majority of cases?

Answer 25

• ACHR, rapsyn and Dok7

Question 26

What is Synapse elimination?

Answer 26

Elimination of excess synapses formed in early development

Question 27

Why is synaptic elimination necessary?

Answer 27

• Many more connections are present during development than are needed (each muscle fibre innervated by multiple motor neurons)
• Excess connections therefore need to be eliminated so that each muscle fibre has a strong connection to one motor neuron

Question 28

What is the mechanism of synapse elimination?

Answer 28

Competition between axons

Question 29

What are the 2 types of axon competition?

Answer 29

Direct - for trophic factors produced by muscle

Indirect - the muscle chooses one axon/synapse over another according to relative synaptic activity at synapse (synapse strength)

Question 30

Evidence shows that which type of synapse competition is the most significant?

Answer 30

Indirect

Question 31

How do differences in synaptic activity cause synaptic elimination

Answer 31

If one synapse is less active than another, the ACHR will be lost
- This causes the loss of the associated synapse

Can be shown experimentally using alpha-bungarotoxin (nicotinic ACH receptor antagonist)