L4: Synapse formation

The assembly of neural circuits can be subdivided into three phases

1. Axonal pathfinding→

   • the projection of axons into the vicinity of poast-synaptic targets

2. Target recognition→

   • cessation of axonal growth upon contact with its postsynaptic targets

3. Synaptogenesis→

   • conversion of growth cone→ specialised pre-synaptic strucutures

   • induction of precisely aligned postsynaptic specialisations in the target cell

Discovery of synapse

• syn→ together

• haptein→ to clasp

• found due to lag time

Features of chemical synapses

• Asymmetric cellular junctions

• Composed of

  1. Presynaptic bouton

  2. synaptic cleft

  3. Postsynaptic specialisation

• control and define turning how many vesciles are released

• depends on the sensitivity of the post synaptic neuron

• relay station

How do synapses form

• Growth cones remodel into synaptic terminals

• How:

  • recognise the site→ mature into synapse

Development of Synapses→ at the presynaptic neuron

Presynaptic

1. Growth cone slows down on approaching targets

2. Filopodial explorations recede and growth cone changes to bouton-like shape

3. Growth cone is aligned with postsynaptic specialisations

4. Vesicle release machinery assembles

5. Growth cone can release neurotransmitter in response to electrical stimulation

Development of Synapses→ at the post-synaptic neuron

Postsynaptic

1. Neurotransmitter receptors cluster opposite presynaptic terminal

2. extra-synaptic transmitter receptors are removed

3. cytoskeletal specialisations form apposed to presynaptic terminal

   • e.g electron dense matrix

Overview of the synaptic development→ what is required

• conversation between the pre and post synaptic neurone

• results in precisely aligned, highly specialised pre ad post synaptic sites

However, these processes can either be:

1. Autonomous→ just always happens

2. Come processes require interactions between pre and post synaptic terminals

next question to ask

• which processes are which

Why is the neuromuscular junction (NMJ) an invaluable model system

• easy access for manipulation

• easy access for observations

• large size

What did observations of synaptogenesis at the vertebrate NMJ show

Clustering of AChRs is indicative of synapse formation

• as the motoneuron growth cone arrives at the target, AChRs begin to aggregate at the site of innervation (presynaptive synaptic site)

How was this shown

1. Use alpha bungarotoxin as a marker for postsynaptic sites

2. binds irreversibly to AChR

3. bound with a fluorescent tag

4. Shows→ growth cones arrive and aggregate into clusters→ forming a synapse

Overall: obervations showed the old ‘axoncentric model of synaptic formation

1. As motor growth cones arrive at the target muscle, AChRs aggregate at the site of innervation (presumptive synaptic site)

2. In addition→ transciption of AChR subunit mRNAs is confined to sub-synaptic muscle nuceli

3. All the while→ pre and postsynaptic cytoskeletal changes occur→ turning the growth cone from an exploratory organelle to a specilaised terminal

4. tranforms the postsynaptic site to an equally specialised region via

   • folding the postsynatpic membrnae

   • secretion of a basal lamina in the synaptic cleft

But where does the synaptogenic signal come from?

The basal lamina of NMJs

How was this found out?

McMahon and Sanes

• denervated or eliminated the target muscle

Result:

• regenerating NMJs reformed precisely as the original sites

• only if the basal lamina was intact

THERFORE

• synaptogenic signal must be from the basal lamina of NMJs

• The extracellular matrix holds information

How was the synaptogenic activity biochemically purified

Procedure:

1. electric organ of torpedo marine ray (essentially a giant cholinergic NMJ) as basal lamina source

2. Assay for synaptogeneic activity for clustering

What was identified:

• Large heparin sulphate proteoglycan→ Agrin

  • Has domains that interact with laminin

• Other synpatogeneic signals from basal lamina

Where was Agrin found to be present

• In motor axons as they made contact with their target muscles

Conformational evidence for agrin’s function

• Knock out in mice

→ Severely impaired NMJ formation

But there are many types of agrins expressed in muscles and in the CNS, which one is used?

• Alternively-spliced isoforms of agrin

How found each one for synaptic formation:

• antibodies for different Agrin isomers

Results

• z-Agrin were essential for synapse formation

So from this evidence, agrin is throught of as…

• the key synaptogeneic signal

• which acts of the growth cone

this was based on mutant phenotypes→ so not completely show the whole picture

Agrin receptor complex at the NMJ

1. Agrin receptor complex

   • with Muscle-Specific Kinase

2. Scaffold formation

Overview of Agrin receptor complex mechanism

1. Agrin binds

2. Activates MuSK

3. activates Lrp4

4. activates HSP90

5. sequesters protease

6. allows cdk5→ bind and anchors down cytoskeleton

7. another kinase cascade

8. break up whole thing??

New idea of the role of Agrin due to other evidence

• Agrin is a maintenance or ‘anti-dispersal’ factor

  • Stabilises neuromuscular junctions

    • by protecting subsynaptic AChR clusters from degradative processes that occur at extra-synaptic sites

What is the evidence for this

1. Zebra fish live imaging→ NMJ formation in zebrafish and of mucules in culture

   • Show: AChRs cluster at presumptive synaptic regions before and also at the absence of motoneuron innervation

2. Approaching motoneuron growth cones preferentially innervate these pre-existing AChR clusters

3. Animals mutant for agrin→

   • AChR clusters also form in the absence of Agrin

4. AChR clusters require Agrin to be maintained→ following motor axon innervation:

   • suggests: motoneurons secrete some AChR cluster dispersal agent (now known to be ACh) which helps to maintain them

1. More info on the Zebra fish live imaging evidence

Procedure:

1. Label with homeobox gene→ labelled the motor neurons with GFR

2. Label AChR receptor in red

   • note: not all labelled to ensure some are still functioing

3. Observe overtime how the AChRs cluster with growth cone approach

Observations:

• growth cones contact receptors and get larger and increase branches to clusters

• post synaptic muscle is guiding the growth cone→ not a passive entitiy

Conclusion:

• AChR clusters precede innervation and are incorporated into newly formed NMJs

• The post synaptic muscle has some pre-patterning of clusters

4. More information→ Evidence that AChR clusters require Agrin to be maintained

Procedure:

• Agrin mutant mouse

• Remove ACh synthesis

Results:

• AChR clusters can form and remain

• Why: because there is no acetly choline to signal AChR cluster dispersal

Conclusion:

• ACh is used as an AChR cluster dispersal factor

4. More in depth experimental evidence about how Agrin maintains ACR clusters

Procedure

• Mice with various mutants

  • no agrin

  • denervated

  • double mutant

Result:

1. No agrin→ AChR clusters can stil form BUT not maintained

2. Denervated→ Clusters are mainted but not innervation

3. Double mutant→ ACh disperses extra-synaptic clusters→ NOT mainatined by nerve derived Agrin

Two conclusions from this

1. Nerves actively dissemble non-symaptic AChR aggregates

2. Simulatenous removal of Agrin and ACh

OVERALL: Agrin maintains the clusters

Therefore this experiment lead to the current view→

1. Postsynaptic pre-pattern→ AChRs clustered before the growth cone arrives

2. ACh from nerve terminal can diffuse beyond the immediate synapse region and lead to dispersal of extra-synaptic AChR clusters

3. Agrin is both an activator of agrin recetptor complex and ‘Anti-dispersal agent’ (maintainence)

1. How is the muscle pre-patterned? (remains controversial)→ Zebrafish evidence

Zebra fish

1. Wnt11r acts as a ligand and activates MuSK on muscles

   • wnt11r from dorso-lateral somites

2. Induces AChR clusters in central muscle region

3. Also guids motor axons along this central muscle region

   • by forming a corridor that is attractive to the growth cones

overall: Wnt11r works as a third party mattch maker for the patterns to match the growth cone approach.→ like how a timetable brings the lectuere and student together without the communication between the two

1. How is the muscle pre-patterned? (remains controversial)→ Mouse evidence

Mouse model→ Wnt-MuSK signalling

1. MuSK kinase being inherent active at low level

2. therefore→ oldest part of the muscle ends up with the greatest MuSK activity

3. therefore→ AChR clustering

Agrin then induces further what

1. Clustering

2. stabilisation

How does it do this

1. Agrin is large and when depoisted on motor axon terminal→ will remain localised in the ECM as the synpase ONLY

2. Activaes Agrin receptor complex

3. Activates on MuSK

4. Activates Lrp4

5. Calpain protease is sequested by Rapsyn (a scaffolding protein)

What does the activation of Lrp4 also do

• Lrp4 also acts as retrograde signal→ inducing presynaptic differentiation

  • helps to align pre with postsynapse

2. How does ACh release casue AChR dispersal

note: ACh is normally blocked by large agrin

1. Dispersal is triggered by AChR mediated clacium influx

2. Activates Calpain protease ( which is normally inactivated by sequestration due to agrin receptor complex activation)

3. activates kinase cascade

4. cdk25 activation by p25

5. promotes dispersal and internalisation of extra-synaptic AChRs

Why is AChR dispersal needed?

• to get rid of extra-synaptic AChR

• ensure that the synapse is precisely aligned

3. How does Agrin work to maintain pre-esxisting AChR clusters (seen above too)

• Locally→ antagonising the ACh dispersal effect

• Globally?→ No

  • extra-synaptic receptor clusters will still be affected by acetyl choline

THEREFORE: this allows for synapse refinement by elination of extra-synaptic sites

The current model for NMJ formation

1. Autonomous synaptic differentiation

• Presynaptic terminal→ Transmitter release machinery

• Postsynamptic terminal→

  1. AChR cluster formation

  2. Postsynaptic cytoskeleton

  3. Sub-synaptic nuclei- specialisation

2. Requirement for pre and postsynaptic signalling

   • Presynaptic terminal→ Cessation of growth and presynaptic differentiation

   • Postsynaptic terminal→

     1. AChR Cluster maintenance

     2. Postsynaptic cytoskeleton maintenance

     3. Precise pre and post synaptic apposition

     4. Structural and functional maturation

The next few cards are on more details of the parts of the Agrin pathway: Downstream of Agrin what happens First stage of synaptic formation

1. Lpr4→ low density lipoprotein receptor-related protein

2. Associates with and signals through a Muscle-specific receptor tyrosine kinase (MuSK)

3. MuSK also binds Wnts

   • In zebra fish→ myotime secreted wnt11r activats MuSK signalling in the central domain of muscles

   • triggers formation of AChR clusters

4. At the same time→ Wnt11r interacts with MuSK present on the grwoth cones of motorneurons

5. Guides them along this central corridor→ towards the pre-formed AChR complexes

Remains to be shown if Wnt-MuSK signalling also peforms such roles in mouse

Second stage→ What happens once motorneuron growth cones have reached muscles…

1. Agrin comes into play

2. Binds to receptor complex

3. MuSK phosphoorylates itself

4. Triggers an intracellular signalling cascade

5. Leads to assembly of te postsynpatic apparatus

thrid stage→ One structrual component is Rapsyn

Postsynapse protein

1. acts as a scaffold

2. mediate interactions between MuSK, AChRs and other postsynaptic cytoskeletal elements

What does the lack of presynaptic specilaisations (e.g MuSK or agrin-deficient mice) suggests

• Presynaptic differentiation requires a retrograde signal from the muscle

What does this retrograde signalling

1. Lpr-4 of the Agrin receptor complex

   • signals retrogradely to induce differentiation of the presynaptic motor axon terminal

2. Signals from basal synaptic lamina→ also induce presynatic specialisations (in regenerating motor axons)

   • e.g basal lamina-associdated Laminin-b2

How is refinement achieved

Dispersal and removal of AChRs from extra-synaptic regions

1. ACh released by motor terminals

2. diffuses beyong regions of synaptically laid down Agrin

3. As no argin→ AChR is exposed to ACh

4. mediate calcium influx

5. activates protease calpain

6. Activates cdk25

Auntonmous vs non-autonomous features of synapse formation

• There is alot of intrinsic signalling between pre and post synaptic partners

But→ some of these happen autonomously and some require interactions between partner cells

• non-cell autonomous

Autonomous processes

1. Muscles→ postsynaptic differentiation in absence of presynaptic motorneurons

2. Presynaptic differentiation→ growth cones appear inherently capabale of releasing neurotransmitter and presynaptic sites form in the absence of poastsynaptic targets

   • Made really wuickly but still really complex→ how is this?

How has it been shwon the presynaptic differentiation is autonomous

• Presynaptic apparatus is assembled from prefabricated modules

• delivered by specialised vesicles

Why is it useful to use prefabicated modules

• facilitates rapid formation of functional release sites

• as soon as presynaptic cell comes into contact with postsynaptic cell

Are post-synaptic specialisations made from pre-fabircated complexes?

• Unclear whether it is pre fabicated or assemble gradually

How do postsynaptic protein complexes assemble in the CNS

• assemble on scaffolding proteins

• containing PDZ domains

• different proteins are responsible for clustering of different neurotransmitter receptors

Example of protein with PDZ domain

PSD-95 protein

• prominent at glutamatergic synapses in vertebrates

Exmaple of specified scaffolding proteins NMJ vs CNS

NMJ

• Rapsyn

CNS

• Gephyrin

  • important for glycine and Stargazin for glutamate receptor clustering

So THEREFORE what is autonomous vs non-autonomous

Autonomous

• Pre and post synaptic specialisation

Non-autonomous

• Location

• Alignment

• Asjustmnet during development

Synapse formation in the CNS→ What happens to the growth cone as synase forms

• changes from highly motile navigational organelle→ presynaptic structure

But what are the signals that induce these changes?

Why is the cerebellum a very successful model system

• regular organisation

• well defined development and connectivity

What are synaptogenic molecules

• Proteins that induce pre or postsynaptic differentiation

  • many of the adhesion complexes can signal bidirectionally

  • some receptors bind secreted factors

Principal types of synapse organising signals

1. Bidirectional organisation

2. Anterograde organizers

3. Retrograde organizers

4. Glial-derived organizers

3. Retrograde organizers found in cerebellum

E.g Frizzled or FGFRs

1. secreted by postsynaptic cells (granule cells)

2. promote differentiation of presynaptic (mossy fibre) growth cones

Examples of these retrograde signals and what they specialise in

• Wnt7A→ but not strictly required as Wnt7a mutant mice still get cerebellar synapses

  • but with a dealy

  • THIS SHOWS→ signals are complex and there are often many of them

In hippocampal CA3 neurons:

• FGF7→ differentiation of inhibitory synapses

  • shown in knockdown in mice→ increased levels of excitation and susceptibility to epilpetic symaptoms

• FGF22→ promotes excitatory synapse

  • knockdown → decrease susceptibility to epileptic symptoms

What was the experimental method for this to be found out

• take granule cell condition medium

• test what induces the fibres to become mossy

  • when come into contact with postsynpatic parters (granule cells)

How we know the FGF7 and FGF22 specilaisaitons:

• can tag them

• see where the spots localise to

But the effect in vivo was not as great→ this showed

• there are multiple signalling things happening

• much more complex interactions

What else is also involved in the formation of excitatory synapses in the CNS

Agrin

Evidence

• Agrin knockout mice show a redcution in excitatory central synapses

More research has shown Argin may be part of a

Coincidence detection system

1. Only when postsynaptic cell’s NMDA receptors are activated

2. Argin is cleaved by the presynaptically secreted argin-specific protease Neurotrypsin

1. Bidirectional organization→ what signals are there

• Neuoligins and neurexins

What do Neuoligins and neurexins do

• induce synaptic differenetiation

  • Neurexin→ post synaptic differentiation

  • Neuroligins→ pre synpatic differentiation

• by aggregation of their trans-synaptic binding partners and scaffold (see image)

• the genes of neurexins cna be spliced into 1000s of isoforms

• forming multiple neuro ligands from just two genes for receptors

HOw do they operate

• Transsynaptic binding partners and scaffolds

• organise post and prescaffold

• anchor cytoskeletal specialisation across the cleft

  • Evidence: only works if you anchor neurorexin moelcules

  • so do not act through signals

What do they do

1. Beads coated with neurexin induce clustering of neurologin

2. Cause postynaptic differentation

and

1. Neuroligin can induce presynaptic differentiation through aggregation of neurexins

2. at the presynatic terminal

overall causes trans-synaptic cell adhesion

What are trans-synaptic cell adhesion molecules

• raft of synamtogenic signals

  • work as pre and post synaptic terminals to specific common ‘meeting regions’

Exmaples of trans-synaptic cell adhesion molecules

• EphB-ephrinB

• SynCAM

One challenge of these trans-synaptic cell adhesion moelcules

• differentiating between what ligand-receptor pairs are capable of under mis-expression conditions

vs

• what aspects of nervous system/synapse development they are required for

• i.e Neurexin and Neuroligin are shown to be capable of inducing post and pre differentiation respectively

What did mutant mice for genes encoding Neuroligin nad neurorexin show

• normal numbers of synapses formed in the CNS

but

• had impaired transmission

Suggests:

• Neurexin and Neuroligin are likely involved in the maturation of synapses

• NOT their induction

A recent study within a population of neurons suggests that the amount of Neuroligin and neurexin may determine…

• how many synapses are formed with each of several partner neurons

→ in a compeitive way

Summary

• synapse formation requires:

  • Exchanges of antero and retro-grade signals

  • between pre and postsynaptic cells

• This communication ensures pre an dpost synaptic specialisation are

  • precise alignment

  • co-ordinated

it is important to separate autonomous and required processes

Summary of Autonomous vs requirment for pre and post synaptic signalling

Autonomous synaptic differentiation:

• Presynaptic terminal:

  • Transport vesicles carry multiple components – “pre-fabricated” complexes.

  • Presynaptic release sites can form in the absence of partner neurons.

• Postsynaptic terminal:

  • Pre-patterns exist in muscles (NMJ), less clear for CNS.

  • Transport vesicles for postsynaptic components ? (controversial – not covered in this lecture)

Requirmnt for pre-postsynaptic signalling

Presynaptic terminal:

• Retrograde signals from the postsynaptic cell induce cessation of growth and promote presynaptic differentiation (Wnt7a; FGF22, FGF7, FGF10).

Postsynaptic terminal:

• Precise pre- & postsynaptic apposition

• Neurotransmitter receptor cluster maintenance Postsynaptic cytoskeleton maintenance