Lecture 21: Synapse Formation

What are the general features of synapse formation in NMJ and CNS?

• synapses are tripartite structures

• cells express most synaptic components before synapse formation (presynaptic terminal, postsynaptic membrane, and glia)

• bi-directional signaling organizes synaptic components at emerging synaptic contacts

• synapses undergo extensive functional maturation

• some of the excess synapses (formed during development) are eliminated

Why is the NMJ referred to has a highly specialized synapse?

• Nerve terminal is capped by a Schwann cell

• nerve terminal is situated in a shallow depression of the muscle cell membrane, which is invaginated into deep and regular post-junctional folds

• AChRs are highly concentrated at the post-junctional fold

Why do we use the central region for pre-patterning for synapse formation?

the central region of the muscle is more mature because myoblasts fuse to developing myotubes at their growing ends

What is required for pre-patterning AChRs?

Lrp4 and MuSK

What is true of Lrp4 and MuSK prior to innervation?

AChRs are concentrated by Lrp4 and MuSK (RTK)

What is true of Lrp4 and MuSK during normal innveration?

• Argin/Lrp4/MuSK signaling stabilizes and stimulates AChR clustering

• ACh alone disperses AChR clustering

AChR clusters are maintained only at nascent synaptic sites

What is true of each myofiber in the synapse?

ensheathed by a basal lamina

What happens in degeneration of muscle, nerve terminals, and Schwann cells?

• MBL and synaptic BL remain

• upon regeneration of muscle, postsynaptic AChRs cluster

What happens during degeneration of muscle and nerve terminals?

• reinnervate by motor neurons

• presynaptic terminal is reformed

What is the synaptic BL signal?

Agrin

• synthesized by motor neurons

• deposited into synaptic BL

• a 200 kDa ptrotein containing 4 EGF-like domains and 3 laminin-G domains in the C-tail, which mediate clustering

What was true of Agrin^-/- mice before and after innveration?

Before: AChRs are initially pre-patterned and stable

After: synapses form transiently but soon are lost. AChR clusters are disperse

What is the signaling pathway for Agrin-Lrp4-MuSK?

1. Agrin binds to Lrp4, which stimualtes Lrp4 interaction with MuSK and activates MuSK

2. Y553 phosphorylation of MuSK recruits Dok7 and Crk

3. Rac and Rho GTPase activation leads to AChR clustering through Rapsyn

4. Synapse-specific transcription likely involve JNK and ETS family TFs

What are the steps in NMJ formation?

1. the growing motor neuron secretes Agrin into the basal lamina

2. Agrin interacts with MuSK in the muscle cell membrane

3. this interaction induced the clustering of ACh receptors in the postsynaptic membrane via Rapsyn

Describe the pre-patterning that requires MuSK (MuSK^-/- mice).

• motor axons fail to stop and differentiate adjacent to the main intramuscular nerve. Instead, axons wander aimlessly over the muscle

• lack AChR pre-patterning

• AChRs and other postsynaptic proteins (Rapsyn) are expressed at normal levels but fail to cluster

Why are MuSK and Agrin required for retrograde signaling to a motor neuron and postsynaptic differentiation?

• MuSK^-/- mice lack normal nervee terminals

• add MuSK back to the muscle, then synapse formation is normal

• Agrin/MuSK activation informs that a functional contact has been made

Why is MuSK required for Agrin-mediated singaling?

• MuSK^-/- or Agrin^-/- mice lack postsynaptic differentiation

• MuSK^-/- or Agrin^-/- mice lack AChR clustering

• dominant negative MuSK blocks Agrin-induced AChR clustering

Why is Rapsyn required for postsynaptic differentiation downstream of MuSK and Agrin?

• Rapsyn is a 43 kDa myristorylated peripheral neuron membrane protein

• binds to AChR (1:1 stoichiometry with AChRs at synaptic sites)

• Rapsyn^-/- moce lack AChR and Dystroglycan but not MuSK clustering

What is Congenital myasthenic syndromes (CMS)?

• caused by mutations in genes encoding for proteins (>30) that are essential for the integrity of neuromuscular transmission

• characterized by fatigable muscle weakness

What is myasthenia gravis (MG)?

• autoantibodies to AChR or MuSK

• characterized by fatigable muscle weakness

What is Lambert-Eaton Myasthenic Syndrome (LEMS)?

• autoantibodies against P/Q type VGCCs on the presynaptic terminal

• characterized by fatigable muscle weakness

What is Spinal muscular atrophy and ALS?

• caused by motor neuron degeneration

Describe the selective gene expression in synaptic nuclei of myofibers.

1. mRNAs encoding AChR subunits at the synaptic sites by local transcription in myofiber synaptic nuclei

2. ETS domain TFs regulate synapse-specific transcription

3. neuronal activity also regulates AChR gene expression

What is true of cells before synapse formation?

they contain most synaptic proteins

What is the steps for the assembly of the central synapse?

1. cells express most synaptic components before synapse formation

2. initial contact through filopodia of a growth cone, and cell surface adhesion/signaling proteins

3. initial contact triggers fusion of APV (active-zone precursor vesicles)

4. deposit active-zone material at the presynaptic membrane

This takes about 10-20 mins

5. synaptic vesicles are recruited to the cell-cell contact site

6. postsynaptic scaffolding molecules, glutamate receptors are delivered to the maturing postsynaptic membrane

   1. NMDAR first then AMPAR

   2. scaffolding proteins (PSD95, NARP, EphB-R, stargazin)

Rapid assembly of synapses within 1-2 hours

What are the steps in formation of a CNS synapse?

1. a dendritic filopodium contacts an axon

2. contact leads to the recruitment of synaptic vesicles and active zone proteins to the presynaptic membrane

3. NT receptors accumulate postsynaptically

What are the synapse-organizing signals at CNS synapses?

1. cell adhesion molecules

   1. Neurexins are presynaptic adhesion proteins that bind to postsynaptic adhesion proteins

   2. Neuroligins and LRRTM2 promote first presynaptic differentiation, and then postsynaptic differentiation

2. Cell surface receptors

   1. postsynaptic neurotrophin receptor TrkC binds to presynaptic receptor tyrosine phosphatase (RPTP) and promotes recruitment of synaptic vesicles to presynaptic sites

How can you investigate different isoforms of neurexins?

• neuroligin can tell and axon to “build a presynaptic terminal here.” by binding neurexin on the axon

• neuroligin binds neurexin, neurexin recruits scaffolds, and the scaffolds bring in vesicles and active-zone proteins. This is bi-directional signaling. The postsynaptic cell instructs the presynaptic side to differentiate.

In the experiment, researchers took HEK293 cells and engineering them to express neuroligin. These were grown alongside real neurons. The synapsin staining marks synaptic vesicles, forming bright clusters where axons cross the green neuroligin-expressing cells.

Expressing neuroligin in a non-neuronal cell is enough to make contacting axons form presynaptic specializations (vesicle clusters and active zones). This happens through neurexin binding, and the presynaptic scaffolds CASK and Mint link neurexin to the vesicles.

A young CNS synapse has NMDARs but no AMPARs. Why is it “silent,” and what makes it functional?

At resting potential, Mg²⁺ blocks the NMDAR pore, so glutamate alone can’t pass current. With no AMPARs to depolarize the membrane, the synapse transmits almost nothing. It becomes functional when activity-dependent AMPAR insertion occurs (AMPARs arrive after NMDARs), with AMPARs held in place by scaffolds like PSD-95/stargazin and NARP.

In postsynaptic development, which glutamate receptor arrives first?

NMDAR then AMPAR

Name two proteins that anchor AMPARs at the synapse, and say how.

Stargazin, which binds AMPARs and PSD-95’s PDZ domain, and NARP, which is secreted and clusters AMPARs extracellularly.

What are the roles of neuronal activity in synapse formation?

• not required for the formation of synaptic structures during development

  • ablation of Munc 18-1 (presynaptic release component)

    • complete loss of synaptic transmission

    • form synaptic structure

• important for synapse maturation and stabilization

  • NARP is induced by neuronal activity

    • clusters AMPARs extracellularly

What is a model mechanism for synapse specificity?

In the hippocampus, dentate granule cells send axons, which form synapses selectively on the dendrites of the pyramidal cells in the CA3 region

• pre- and postsynaptic partners express the same homophilic adhesion molecule (Cadherin-9) and thereby preferentially form synapses with each other