Lecture 21: Synapse Formation

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

• synapses are tri-partite structures (presynaptic terminal, postsynaptic membrane, and glia)

• cells express most synaptic components before synapse formation

• 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

What are the three components of a tripartite synapse?

• presynaptic terminal

• postsynaptic membrane

• glia

What caps the nerve terminal at the NMJ?

a Schwann cell

Where is the nerve terminal located relative to the muscle membrane?

in a shallow depression of the muscle membrane

What are post-junctional folds?

deep regular invaginations of the muscle membrane

Where are AChRs highly concentrated?

at post-junctional folds

Why is the central region of the muscle the location for muscle pre-patterning?

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

What happens to AChR clusters following innervation?

synaptic signaling stabilizes them

Which proteins are required for AChR pre-patterning?

Lrp4 and MuSK (muscle-specific kinase)

What type of receptor is MuSK?

a receptor tyrosine kinase (RTK)

What does Agrin/Lrp4/MuSK signaling do?

stabilizes and stimulates AChR clustering

What effect does ACh have on AChR clustering?

ACh alone disperses AChR clusters

Why are AChR clusters maintained only at nasvent synaptic sites?

because Agrin/Lrp4/MuSK signaling promotes clustering while ACh disperses clustering elsewhere

What differentiation signals are contained within the synaptic basal lamina?

signals for both presynaptic and postsynaptic differentiation

What is the muscle basal lamina (MBL)?

a basal lamina that ensheaths each myofiber

What happens when muscle, Schwann cells, and nerve terminals degenerate but synaptic basal lamina remains?

postsynaptic AChR clusters reform

What happens when motor neurons reinnervate the preserved synaptic basal lamina?

presynaptic terminals reform

What is the critical signaling molecule in the synaptic basal lamina?

Agrin

What is Agrin synthesized by? Where is Agrin deposited?

• synthesized by motor neurons

• deposited into synaptic basal lamina

What is Agrin’s molecular weight, and how many EGF-like domains does Agrin contain?

• 200 kDa protein

• contains 4 EGF-like domains

How many laminin-G domains does Agrin contain? Where are Agrin’s laminin-G domains located?

• three domains

• located in the C-terminal tail

Which Agrin domains mediate clustering?

laminin-G domains

What is observed in Agrin KO mice before innervation?

AChRs are initially pre-patterned and stable

What happens to synapses after innervation in Agrin KO mice?

they form transiently and are then lost

What happens to AChR clusters after innervation in Agrin KO mice?

they disperse

List the steps in the Agrin-LRP4-MuSK signaling pathway in NMJ synapse formation.

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

2. Y553 phosphorylation of MuSK recruits Dok7 and Crk

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

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

List the steps in the formation of a NMJ.

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

2. Agrin interacts with MuSK in the muscle cell membrane

3. This interaction induces the clustering of ACh receptors in the postsynaptic membrane via rapsyn

MuSK is located in the muscle, but somehow MuSK activation signals back to motor neuron to stop wandering. How?

through Lrp4-MuSK

What happens to the motor axons in MuSK -/- mice?

motor axons fail to stop and differentiate adjacent to the main intramuscular nerve; axons wander aimlessly over the muscle

What do MuSK -/- mice lack?

AChR pre-patterning

In MuSK -/- mice, AChRs and other postsynaptic proteins (rapsyn) re expressed at normal levels but fail to what?

cluster

Overall, what is defective in MuSK -/- mice?

pre and postsynaptic differentiations

MuSK and Agrin are required for what type of signaling to the motor neuron?

retrograde signaling

MuSK and Agrin are required for what aspect of presynaptic development?

presynaptic differentiation

What presynaptic phenotype is observed in MuSK -/- mice?

MuSK -/- mice lack normal nerve terminals

What experiment demonstrates that MuSK in muscle is required for synapse formation?

adding MuSK back to the muscle restores normal synapse formation

What does Agrin/MuSK activation communicate to the motor neuron?

that a functional contact has been made

MuSK is required for what type of signaling?

Agrin-mediated signaling

What hapens to postsynaptic differentiation in MuSK -/- mice?

postsynaptic differentiation is absent

What do MuSK -/- and Agrin -/- mice have in common regarding postsynaptic differentiation?

both lack postsynaptic differentiation

What happens to AChR clustering in MuSK -/- mice?

AChR clustering is absent

What do MuSK -/- and Agrin -/- mice have in common regarding AChRs?

both lack AChR clustering

What is the effect of dominant-negative MuSK?

it blocks Agrin-induced AChR clustering

What does the dominant-negative MuSK experiment demonstrate?

MuSK is required for Agrin-induced AChR clustering

Rapsyn is required for what developmental process?

postsynaptic differentiation

Rapsyn functions downstream of which two molecules?

MuSK and Agrin

What is the molecular weight of Rapsyn?

43 kDa

What post-translational modification is present on Rapsyn?

myristylation

What type of membrane protein is Rapsyn?

a peripheral membrane protein

What does Rapsyn bind?

acetylcholine receptors (AChRs)

What is the stoichiometry between Rapsyn and AChRs at synaptic sites?

approximately 1:1

What happens to AChR clustering in Rapsyn -/- mice?

AChR clustering is absent

What happens to dystroglycan clustering in Rapsyn -/- mice?

dystroglycan clustering is absent

What happens to MuSK clustering in Rapsyn -/- mice?

MuSK clustering remains intact

What does the Rapsyn -/- phenotype indicate the relationship between MuSK and Rapsyn?

MuSK clustering can occur without Rapsyn, placing Rapsyn downstream of MuSK

Which molecules are required for retrograde signaling to the motor neuron?

MuSK and Agrin

Which molecules are required for postsynaptic differentiation and AChR clustering?

• MuSK

• Agrin

• Rapsyn

Which clustering is preserved in Rapsyn -/- mice?

MuSK clustering

Which clustering is lost in both MuSK -/- and Agrin -/- mice?

AChR clustering

Which clustering is lost in Rapsyn -/- mice?

AChR and dystroglycan clustering

What is congenital myasethenic syndromes (CMS) caused by?

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

What is CMS characterized by?

fatigable muscle weakness

What is the cause of myasthenia gravis (MG)?

autoantibodies to AChR or MuSK

What is MS characterized by?

fatigable muscle weakness

What is Lambert-Eaton myasthenic syndrome (LEMS) caused by?

autoantibodies against P/Q type VGCCs on the presynaptic terminal

What is LEMS characterized by?

fatigable muscle weakness

What is spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) caused by?

motor neuron degeneration

How do AChR clusters in the central region of the muscle prior to innervation?

motor axons approach and form synapses in the prepatterned region of the muscles where AChR transcripts and proteins are

Describe the selective gene expression in synaptic nuclei of myofibers.

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

• ETS domain transcription factors regulate synapse-specific transcription

• neuronal activity also regulates AChR gene expression

True or False: CNS synapses are tripartite junctions.

true

True or False: Cells contain most synaptic proteins before synapse formation.

true

What signaling organizes synaptic components at emerging synaptic contacts?

bi-directional signaling

True or False: Synapses undergo extensive functional maturation and elimination.

true

What are the steps of central synapse assembly?

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

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

6. postsynaptic scaffolding molecules, glutamate receptors are delivered to the maturing postsynaptic membrane [NMDAR first then AMPAR and scaffolding proteins PSD95, NARP, EphB-R, stargazin)

How long does it take for presynaptic terminals to form?

10-20 minutes

True or False: There is rapid assembly of synapses within 1-2 hours.

true

List the steps in the 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. neurotransmitter receptors accumulate postsynaptically

What are the cell adhesion molecules in the CNS synapse?

• neurexins

• neuroligins

• LRRTM2 (leurine-rich repeat protein 2)

What are the presynaptic adhesion proteins that bind to postsynaptic adhesion proteins?

neurexins

Which proteins promote first presynaptic differentiation and postsynaptic differentiation?

neuroligins and LRRTM2

Describe cell surface receptors at the CNS synapses.

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

True or False: There are 4000 different isoforms of neurexins provides diversity and specificity of synapse formation.

true

How do you investigate the neurexin and neuroligin interactions for presynaptic development?

co-culture neurons and HEK293 cells expressing neuroligin

What is the hallmark of postsynaptic development?

accumulation of postsynaptic glutamate receptors

Describe the accumulation of postsynaptic glutamate receptors.

• NMDAR first then AMPAR

• scaffolding proteins (NARP, EphB-R, stargazin, membrane associated guanylate kinase (MAGUK) scaffold proteins including PSD95)

True or False: The PSD of human, mice, and other mammals comprises 1500 proteins. The total complement of synaptic proteins is around 2000 proteins.

true

True or False: Mutations in over 200 genes encoding PSD proteins in humans result in over 130 brain diseases.

true

Aberrant synapse physiology may contribute to a variety of disorders such as?

• neurodevelopmental (autism, down syndrome)

• neurological disorder (epilepsy)

• mental retardation

• neuropsychiatric disorder

• neurodegenerative disorders (Alzheimer and Parkinson’s disease)

What is the role of neuronal activity in synapse formation?

• not required for the formation of synaptic structures during development

• important for synapse maturation and stabilization

What does ablation of Munc 18-1 (a key presynaptic release component) lead to?

• complete loss of synaptic transmission

• form synaptic structure

What induces NARP?

neuronal activity

True or False: Synapse maturation and stabilization clusters AMPARs extracellularly.

true

In the hippocampus, dentate granule cells (DF) send axons (mossy fibers) which form synapses selectively on the dendrites of the pyramidal cells in the CA3 region. How does this occur?

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