Synapses Exam 4
CNS neurons receive...
100s of inputs that cover the majority of the neuron
Adult AChR form
2 alpha, 1 beta, 1 epsilon, 1 sigma
Embryonic AChR form
2 alpha, 1 beta, 1 gamma, 1 sigma
How many sites of phosphorylation does adb have?
3
Extracellular domain of cadherins
5 ectodomains that mediate Ca binding
C6Box
6 phylogenetically conserved cysteine residues in MuSK
What have also been involved in synapse formation?
Synaptic adhesion like molecules, SALM1 and 2
What are diffusible factors synthesized by?
Target neurons or glial cells
Mechanism of synapse elimination is via...
Terminal branch loss
Possible model for AChR clustering
Untethering of affected AChRs from the cluster site could lead to a lower AChR density and prevents accumulation of AChRs
Activity dependent mechanisms of development
take place once the axons of neurons are connected to other neurons, influenced by the enviornment
What makes myotubes completely unresponsive to agrin?
MuSK -/-
What happens when agrin is added to myotubues?
MuSK is rapidly activated and induces the formation of new receptor clusters
What else did the experiment with separated growth cone and muscle show?
Muscle fibers have AChR before innervation by motor neurons
Conditional Mutagenesis
Mutant phenotype only expressed under extreme enviornments
Both synapses are blocked:
Neither is punished
Formation of nascent AChR clusters at E16.5-18.5 is induced by...
Nerve provides agrin and ACh as + and - signals to regulate synaptic connections
Formation of nascent AChR clusters at E14.5 is induced by...
Nerve-independent, muscle-intrinsic and MuSK-dependent mechanism
Molecules involved in growth cone guidance
Netrins, semaphorins, ephrinA
What does overexpression of SALM1 promote?
Neurite outgrowth of young neurons and co-clusters with PSD95 and NMDAR
Experimental Design for Fibroblast growth factor and synaptogenesis
Neurons were treated and purified with FGF22 and immunostained with antibodies against neurofilments, synapsin and SV2.
Does PNS synapse integrate the response of inputs?
No
Is agrin required for initiation?
No
Is the 'loser' of synapse elimination dependent upon surface area?
No
Was dissipation seen outside of the illuminated region?
No
Rapsyn null mice
No AChR clusters at all on the muscle
What did mice deficient in MuSK show?
No detectable signs of postsynaptic differentiation (despite normal levels of AChR expression)
What is the evidence for MuSK being one subunit to a multisubunit receptor?
No direct binding of purified agrin to either purified MuSK, with other subunits being either necessary or solely responsible for agrin binding
Erbb-/- NMJs
No obvious abnormalities -No differences in levels of epsilon AChR, sigma AChR and MuSK mRNAs in synaptic and extrasynaptic regions -Showed that there were not in vivo effects of neuregulin only in vitro
Synapse Elimination when both synapses are protected and active:
No punishment
Development of synaptic defects in E17 - agrn -/-
No synapses/colocalization of nerve and muscle. Clusters dispersed.
Was there a difference between adding neuregulin to adb mutants?
No! No effect of neuregulin on adb
Effect of neuregulin without adb?
None
What kind of dose of BTX should be used for labeling purposes?
Not full saturation. Need to be careful not to block activity.
Is neuregulin R not part of dystrophin protein complex?
Not part of the complex
Mutant alpha-dystrobrevin
Fewer receptors in the mutant, more susceptible to disease. Mutate 3 phosphorylation sites
What domain is required for agrin responsiveness?
First Ig-like domain
Clusters in which only rapsyn-GFP was bleached
Fluorescence rapidly recovered
Alexa-594 mediated dissipation only occurred if...
Fluorophore was relatively close to the AChR, not conjugated to a secondary aB
Co-expression of AChRs and rapsyn in any cell
Formation of AChR-rapsyn co-clusters
How was it found that growth cones release NT without a synapse?
Found out by recording from a muscle that is next to the growth cone and far from the growth cone
Ectodomain of MuSK
Four immunoglobulin-like domains Ig-1 to Ig-4 and a region with 6 conserved cysteine residues
What do WNTs bind to?
Frizzled receptors and also act directly on cytoskeletal organization
Myotubes are formed from
Fused myoblasts
Mice that lack neural agrin but have MuSK-L/H transgenes
-Survive for weeks -MuSK transgenes can restore functional synapses
Results of glia astrocytes increase the number of puncta containing both pre/postsynaptic proteins
Absence of glia: synaptotagmins are diffuse throughout the cell and few PSD-95 puncta Presence: Synaptotagmins are discretely clustered and there are lots of PSD-95 puncta
Possible explanation for Alexa-594 induced dissipation
Absorption of photons by fluorophore causes structural modification of AChR.
What do WNTs act through?
Activation of beta catenin
What do all of the KO/experiments with agrin show?
Agrin is a key component of receptor clustering
What does mice without agrin but with MuSK-l/H suggest?
Agrin is nonessential for receptor clustering and prepatterning`
3rd Posible role of neuregulin in postsynaptic differentiation
Agrin might direct an autocrine pathway in which muscle-derived neuregulins potentiate or mediate effects on AChR transcription.
When receptors were induced to disperse what happened to scaffold proteins?
All proteins were removed from cluster and new weren't accumulated
In the vertebrate nervous system in what manner do the developmental processes occur?
Almost simultaneously
What is large and what is small, alpha neurexin vs beta neurexin
Alpha is large, beta is small
Prepatterning Normal
At E18 receptors cluster in the middle of the muscle
Glial diffusible factors
Cholesterol, thrompospondin
Development of synaptic defects in E17 - DKO
Clusters maintained.
Neuregulin binding to receptors...
Controls alpha dystrobrevin, critical component for stability of Rs. Neuregulin can indirectly control the stability of receptors at the synapse
CdK5
Cyclin dependent kinase. A thr/ser cytoplasmic kinase. Highly expressed during muscle development.
What blocks agrin signaling?
Dominant negative MuSK
Neuregulin
Interacts with erbB kinases to induce selective expression of AChR subunit genes by synaptic nuclei
What does the results from absence of prepatterning in rapsyn mutants indicate?
Involved in the confinement or the guidance of motorneuron terminals to the center of muscle fibers
N-Cadherin
One of first proteins to localize the newest synapses. Important for neurite outgrowth, guidance and synapse formation
Results of test whether cholinergic agonists disperse AChR clusters in the absence of agrin
Only carbachol and ACh induced dispersion of AChR clusters. Agrin antagonized ACh-induced dispersion of AChR clusters. -An increase in clusters indicates more dispersion.
What are inducers involved in?
Postsynaptic clustering
What happens to agrin deficient mutant mice?
Postsynaptic differentiation is profoundly impaired
What blocks effect of WNT-3?
Preincubation of secreted frizzled-related protein-1 with WNT-3
Neuregulin Receptors
Tyrosine kinases (ErbB kinases) Concentrated at postsynaptic membrane at NMJ
Is MuSK required for AChRs clustering? How?
Yes. Fluorescently labeled nerves with and without MuSK and AChRs and overlapped them
How does agrin signaling work?
Z+ agrin binds to its receptor and initiates the aggregation of AChRs
What is the only spliced form of agrin?
Z+, z- can be formed in vitro
Synapse formation involves the pairing of...
pre and postsynaptic partners at a specific location
Chromophore-Associated Light Dissipation
AChRs labeled with Alexa 594 and exposed to laser light was dissipated from clusters on myotubes and prevented accumulation of new AChRs
What is responsible for positive and negative signals for synapse formation at NMJ?
+ - Agrin - - ACh
What did this dissipation experiment show?
-AChR necessary for rapsyn clustering in mytotubes -threshold density of AChRs provides a signal that allows proteins of the DG complex and intracellular scaffold to stay clustered -This allows insertion of new proteins
Results from Testing the Role of Postsynaptic Activity in Synapse Elimination
-Active - winner, inactive - loser -Requires a balance of synaptic activity. -For elimination - one must be active and one must be inactive.
What evidence is there that synapse elimination occurs?
-2 NMJs imaged over 3 days. -Transgenic mice with YGFP in motonuerons and AChRs labeled with BTX -Day 7: NMJ1 multiply innervated, NMJ2 is singly innervated -Day 8: One axon in NMJ1 becoems smaller -Day 9: Thin branch is eliminated
Extrasynaptic Repression
-ACh represses AChR subunit gene expression in non synaptic nuclei
Results for cell death as reason for synapse elimination?
-After induced cell death there was labeling but it was the cells that were killed. Not additional -No evidence of cell death at the time of synapse elimination
What is seen in synapses around P8 after birth?
-Before elimination there is segregation -A separation of motoneurons
What is induced with binding of EphrinB to EphBR?
-Bidirectional signaling between the receptor and ligand containing cells -Organization of pre/postsynaptic glutamatergic terminals
Experimental Procedure to determine whether cholinergic agonists activate Cdk5 to induce the disassembly of AChR clusters
-C2C12 myotubes treated with carbachol, agrin or both -Lysates IP with anti-Cdk5 aB and subjected to kinase assay using H1 as substrate -Level of phospho-H1 was used to indicate Cdk5 activity
Evidence for loss of long axon collaterals of axons erroneously projecting to multiple muscles...
-Cholera toxins with fluorescent labels injected into two different muscles at P0 and P14 -Dyes traveled to spinal cord -Wanted to see if doubly labeled neurons were present in spinal cord
Protocadherins
-Class of cadherins primarily expressed in the nervous system -Weak adhesive properties -Unknown function
Cadherins
-Classical Adhesion molecules -Ca dependent -In both pre/postsynaptic compartments -Form strong synaptic adhesions
Genetics Inactivation of the Cdk5 Gene Maintains AChR clustering in AGD Mutants
-Diaphragm from Cdk5 KO and AGD/Cdk5 double KO immunostained with BTX-R and synaptophysin-G -No AChR clusters in AGD -AGD/Cdk5 KOs AChR clusters rescued
Experiment to test whether inhibition of cholinergic ACh production in AGD mutants may lead to maintenance of AChR clusters that would have otherwise dispersed
-Double KO in AGD and ChAT -Fluorescent analysis of AChR clusters
Results to test role of Cdk5 with agrin
-Few AChRs present in agrin mutants -Lots of AChR clusters present in agrin mutants treated with Rosc
What happens after start of NMJ development?
-Growth cone and surface of myotube contact and ACh is detected in the muscle cell -Pre and postsynaptic differentiation (formation of active zone and receptor clusters)
What can clustering and binding of adhesion proteins do?
-Initiate the recruitment of synaptic proteins -Lead to the activation of intracellular signaling events -Lead to the induction of dendritic spine formation
What is the evidence for age-related changes in the NMJ?
-Label receptors with BTX -Label presynaptic terminals -Analyze fluorescence changes over aging periods
Why did most of our knowledge about synaptogenesis come from the NMJ?
-Lots of similarity -Large size -Simple -Easily accessible -High density of receptors -Availability of BTX that binds specifically and irreversibly to AChRs
Experimental procedure to test role of Cdk5 with agrin
-Mice deficient in agrin and WT injected with Rosc at E14.5 -Embryos collected at E16 and AChR clusters in diaphragm with fluorescence analysis using BTX-F
Evidence for cell death as reason for synapse elimination?
-Motor axon labeled with caspase 3 and aB for caspase 3 -No labeling in the motor neurons -Induced death and then saw labeling
Absence of prepatterning of AChRs in Rapsyn mutants
-Motor axons undifferentiated and grow into non-synaptic regions
Description of synapses at birth
-Multi-innervated -Axons enter in an intertwined bundle from one direction
Experimental procedure to show NRG/ErbB signaling phosphorylates aDB
-Myotubes from erbb2/4 -/- myoblasts -IP with a-syntrophin -Immunoblot with anti-phosphotyrosine (4G10) aB and aDB aB.
Experiment to test whether NRG/ErbB phosphorylates tyrosine residues in alpha-DB1
-Myotubes incubated with ErbB blocers or NRG1B in presence or absence of perovisanadate -Lysates immunoprecipitated with an antibody recognizing syntrophins -Precipitates denatured and blotted with anti aDB1 and anti-syntrophin
How are CNS and PNS synaptic clefts different?
-NMJ has a well developed basal lamina -CNS doesn't show a detectable basal lamina
Agrin and Lrp4
-Neural agrin can bind directly and selectively to lrp4 with high affinity -Agrin binding increases its interaction with MuSK, which increases its activation via dimerization
Results for loss of long axon collaterals of axons erroneously projecting to multiple muscles...
-No evidence of doubly labeled neurons at either dya -Few labeled cells due to small amount of dye spill over between nearby muscles
Results of test whether inhibition of cholinergic ACh production in AGD mutants may lead to maintenance of AChR clusters that would have otherwise dispersed
-Numerous AChR clusters present in AGD/ChAT double mutants. Lack of ChAT rescued the AGD in postsynaptic maturation -AGD no synapses at all -ChAT -/- more clusters
Results to determine whether cholinergic agonists activate Cdk5 to induce the disassembly of AChR clusters
-Phospho-H1 levels increased following AChR agonist carbachol treatment -Carbachol treatment increases Cdk5 kinase activity -Rosc inhibited carbachol induced Cdk5 activity
NMJ of double KOs of AGD and ACh
-Postsynaptic AChR clusters missing in agrn -/- -Present in widened end-plate band in both chat -/- and chat&agrn -/-
What is netrin-G2 ligand?
-Postsynaptic cell surface protein that instructs the formation of excitatory synapses -Promotes assembly of postsynaptic scaffolding molecules and receptors
What kind of receptor is MuSK?
-Receptor tyrosine kinase specific for muscle cells -expressed and concentrated in the postsynaptic membrane of the skeletal muscle
SALM2
-Regulates formation of synaptic sites and clustering of AMPARs -Regulates organization of postsynaptic terminal but not the presynaptic
Phenotype of ErbB3/4 mutants
-Severe neuromuscular defect -Some transcriptional specialization of synaptic nuclei occurs
Musk
-Studies suggest that Musk is the receptor for agrin -Selectively activated by z+ agrin, aggregates AChRs
Agrin and AChR Clustering
-Sufficient to induce ectopic AChR clusters in adult muscles -Induces formation of a postsynaptic apparatus in denervated muscles -Induces the formation of AChR clusters on the surface of cultured myotubes
Similarities between PNS and CNS synapses?
-Terminals stick to specialized postsynaptic apparatus -Capped by glial cell -Synaptic vesicles concentrated at release sites -Same vesicle proteins -Synaptic release vesicles are the same -Postsynaptic apparatus has high [receptor] -Specialized cytoskeletal apparatus -Synaptic cleft -Clustering, removal, and recycling of receptors are similar -Electrical activity regulates receptor levels -Switch in receptor subunit during development -Synapse elimination
What is the evidence that the eliminated synapse isn't always the smaller neuron?
-Transgenic mice with spectral variatns in motor axons. Monitored axons at developing NMJs. -Larger motorneuron was eliminated
Results that NRG/ErbB phosphorylates aDB
-Tyr p-lation of adb undetectable w/ or w/o NRG1B -ErbB blockage caused abolishment of aDB p-lation -Not due to nonspecific ErbB pharmacological blockers
Results of test whether NRG/ErbB phosphorylates tyrosine residues in alpha-DB1
-Untreated cells->substantial phosphorylation of a-DB1 and addition of NRG1B didn't further increase phosphorylated aDB -Block of NRG/ErbB reduced phosphorylation of adb
Experimental Set Up of Testing the Role of Postsynaptic Activity in Synapse Elimination
-Used adult mice -Labeled muscle with a low dose of BTX and a dye that labels vesicles -Added high concentration of BTX to one specific area of the synapse to block transmission and imbalance of synaptic activity -After 4 weeks, found same synapse and imaged again. BTX injection was eliminated -Then blocked the entire synapse, now the synapse stayed the same.
Results to prove for neuregulin/ErbB stabilizing agrin-induced AChR clusters via phosphorylation of alpha-DB1
-WT myotubes with anti-NRG1B show reduced agrin-induced AChR clusters vs myotubes with NRG1B -AChR clusters in mutants aren't affected by NRG1 neutralization or addition of NRG1B -Stability of AChR cluster could be rescued when a WT construct of a-DB1 was expressed in mutant myotubes (phosphorylatable)
Experiment to prove for neuregulin/ErbB stabilizing agrin-induced AChR clusters via phosphorylation of alpha-DB1
-WT myotubes, adb-/- mutant, and mutants transfected with GFP-adb1 -Add agrin to cluster receptors -Treated with function-blocking aB to neutralize NRG1Beta activity secreted by myotubes -Cells treated with either NRG1B or fresh anti NRG NRG1B -Cells bathed with fluorescent BTX to label AChR clsuters
Why is Cdk5 a great candidate for negative signals that disperse AChR?
1) Activation reduces clustering of PSD95 and ion channels 2) Cdk5 deficient neurons show increased PSD cluster size
What three signaling molecules does the nerve terminal release?
1) Agrin 2) Neuregulin 3) ACh
WNT biochemical pathway
1) Bind to frizzled 2) Activate dishevelled 3) Activate GSK-3/Axin/APC 4) Activate B-Catenin 5) B-Catenin enters the nucleus and activates gene expression
Synapse elimination four step overview
1) Birth of neurons 2) Outgrowth of axons and dendrites 3) Synaptic connections made 4) Refinement of synaptic connections
What are the three distinct mechanisms might contribute to the high density of AChRs at the NMJ?
1) Clustering of diffusely distributed AChRs in the postsynaptic membrane 2) Transcriptional activation of AChR subunit genes in subsynaptic nuclei 3) Transcriptional repression of AChR subunit genes in nonsynaptic myonuclei
What are some age-related changes in the NMJ?
1) Complete denervation of postsynaptic AChR site 2) Fragmentation and faintly labeled patches of receptors 3) Innvervating axons are thin and have swellings near the terminal and sprouting
Genetic tricks to avoid embryonic lethality
1) Deletion of neuron-specific isoform 2) Conditional Mutagenesis
Four possible mechanisms of synapse elimination are...
1) Late motorneuron death 2) Loss of long axon collaterals of axons erroneously projecting to more than one muscle 3) Loss of long axon collaterals within a muscle 4) Pruning of many terminal branches of axons within the target field
Procedure for Alexa-594 Mediated Dissipation of AChR
1) Myotubes incubated with fluorescently labeled BTX 2) Illuminated with laser 3) Incubated with different fluorescently labeled BTX 4) Imaged to see new receptors
Procedure for Complete Dissipation of the AChR Scaffold
1) Myotubes labeled with BTX-Alexa-594 2) Individual clusters were illuminated 3) Myotubes fixed and labeled with anti-AChR aB and either anti-rapsyn, anti-DG, anti-Utr, anti-actin or Phalloidin FITC
Procedure for Laser-Induced AChR Dissipation
1) Myotubes transfected with rapsyn-GFP and labeled with BTX-594 2) Clusters imaged and illuminated either to bleach GFP or excite Alexa-594 3) Cells fixed and labeled with anti-AChR aB
2 opposing activities promote synapse formation:
1) Positive signals stabilize innervated AChR clusters 2) Negative signals disperse those that aren't innvervated
Inductive factors
1) SynCAM 2) Neurexins/neuroligins 3) EphrinsB/EphrinsBR
Two steps of synaptogenesis
1) Synaptic specificity 2) synaptic assembly
Steps of Mechanism of Synapse Elimination
1) Synchronous release of ACh 2) Local Protection Cloud 3) Spreading of punishment 4) Loss of synapse maintenance factor 5) Axon atrophy and withdrawal 6) Can be spread to other synapses, but diffuses as it goes along.
Three molecules involved in specification and induction of synapse formation
1) diffusable factors 2) CAMs 3) Inducers of synapse formation
Two groups of diffusible factors:
1) neuronal factors 2) glial factors
What kind of signals to muscles receive?
A single, massive input at a single sites so most of their surface is non synaptic
Connections between AChR, DG, Utr, rapsyn and actin
AChR binds rapsyn Rapsyn binds DG DG binds Utr Utr binds actin
Prepatterning
AChR clusters in the middle of the muscle guides motorneuron growth cones
Clusters in which Alexa-594 illuminated
AChR induced to dissociate and rapsyn-GFP decreased over time
Lrp4 Mutants
Baby mice were smaller than wt. Didn't have synaptic clustering at E18.5
SynCAM1
Best studied and Ca independent. Regulates number of presynaptic specializations
What is extrasynaptic repression mediated by?
Binding of myoD-family transcription factors to E-box sequences in AChR gene promoters
Where is agrin synthesized?
By motor neurons, transported down motor axons and released from nerve terminals, where it stably associates with the basal lamina of the synaptic cleft
Experiment to test whether cholinergic agonists disperse AChR clusters in the absence of agrin
C2C12 treated with agrin overnight to induce AChR clusters. Then washed and added either agrin, carbachol, or both. Visualized with BTX-R.
What are two adhesive factors?
Cadherins and protocadherins
What do neurexin and neuroligins do?
Can induce formation of excitatory and inhibitory synapses. Act as Ca dependent cell adhesion molecules
What does expression of dominant negative cadherins do?
Causes perturbation of presynaptic markers and postsynaptic scaffold proteins
Remove phosphorylation sites of adb?
Decreased clustering
What can increase the density of extrasynaptic receptors?
Denervation. Increases transcription of AChR subunit gene by extrasynaptic nuclei. Reversible process.
What happens during cell fate specification?
Development of distinct morphology, axonal and dendritic trajectories
Mice deficient in neuregulin or ErbB2
Die at early embryonic stages, can't asses phenotype
How can synapse fragmentation be prevented?
Diet and exercise
Postsynaptic scaffold organization
Discrete units of intracellular scaffolding proteins associated with individual receptors vs matrix of cross-linked proteins
Cdk5-dependent activity does...
Disperses AChR clusters that aren't apposed by nerve terminals
What did a lack of antibody staining indicate?
Dissipation of intracellular scaffold and prevention of new scaffolding protein insertion
1st Mechanism of synapse assembly
Distribution of receptors from primitive clusters to the synaptic area via lateral movement, diffusion in the plasma membrane or endo/exocytosis
WNT pathway has __________ cellular responses in both embryonic development and in adults
Diverse
Prepatterning with agrin KO
E18 no receptors at all
HAS-MuSK-H (OE)
E18 receptors all over the muscle. Dictates the pattern of AChR clustering
Why is having more than one synapse per NMJ bad?
Each AP will fire and have non-synchornization of APs. You end up with constant firing and causing tremors. Important for synaptic specificity.
What happens when agrin is introduced into denervated muscles?
Elicits formation of a remarkably complete postsynaptic apparatus
Experimental Design of WNT-3 Inducing Axonal Remodeling in Sensory Neurons
Embryonic sensory neurons cultured with or without WNT-3. Immunostaining for GAP-43, which helps visualizing neuronal morphology
Dok-7
Essential for neuromuscular synaptogenesis in vivo
What happened to cultured HC neurons from mice lacking EphB1-3?
Exhibit complete loss of excitatory postsynaptic specialization and dendritic spines
What starts the development of the NMJ?
Expression and release of ACh from growth cones
Result for Fibroblast growth factor and synaptogenesis
FGF22 induces vesicle clustering and neurite branching. Increase in number of synaptic clusters per neurite
AChR-deficient muscles, C2C12 myotubes without AChR, mice lacking adult epsilon AChR subunit...
Fail to form mature synapses without rapsyn clustering
Development of synaptic defects in E13 - agrn -/- and ChAT -/-
Grows extensively no limit. Shows that AChR clustering is independent of both agrn and ACh at this time.
Cytoplasmic region of MuSK
Has a recognition site NPXY for phosphotyrosine binding domain-containing proteins as well as the ATP binding/kinase domain are both essential for activity
What structural characteristics to neurexins and neuroligins have in common?
Highly glycosylated region, transmembrane domain, and terminate in PDZ domain binding sites
What was BTX used initially to do?
ID, purify and track receptor movement
What are CAMs involved in?
Inducing the assembly of pre and postsynaptic specialization
Rosc
Inhibitor of Cdk5
Perovixanadate
Inhibitor of phosphatases
How would you introduce a molecule into a muscle?
Injection of an expression vector or the recombinant protein itself
Agrin
Large heparan sulfate proteglycan that interacts with MuSK/LRP and aggregates rapsyn mediated AChR clustering
Intracellular domain of cadherins
Linked to the cytoskeleton via catenin molecules
Syntrophin
Localize signaling proteins because it is apart of dystrophin protein complex
Agrin-dependent activity does...
Locally stabilizes AChR clusters apposed by nerve terminals
LRP General
Low density lipoprotein receptor related protein. Roles in lipid metabolism, cholesterol homeostasis and Wnt signaling. Many have specific roles in development of nervous system
ACh
Major excitatory NT at NMJ. Synthesized by ChAT. Activates muscles in PNS.
Synaptophysin
Marker of presynapstic vesicles
Dok-7 dysfunction
May be involved in pathogenesis of NMJ disorders
Rapsyn
Membrane-associated cytoplasmic protein
What germ layer is muscle tissue formed from?
Mesoderm
Neuronal diffusible factors
Molecules involved in growth cone guidance, Wnts, fibroblast growth factors
What induces cell fate specification?
Morphogens and transcription factors
Classical view of NMJ formation
Motor axon approaches a newly formed myotube and secretes a factor that organizes postsynaptic differentiation at the site of contact
Why would they stain synaptotagmin and PSD-95?
Presynaptic and postsynaptic markers!
Non-neuronal cells expressing EphB2 trigger...
Presynaptic differentiation of contacting axons in co-cultured neurons
Activity independent mechanisms of development
Proceed according to genetic programming independent of the enviornment
Synaptic Assembly
Process that directs how synapses are formed: from the assembly of the presynaptic structure to the formation of the postsynaptic specializations
Synaptic specificity
Process that directs where synapses form: from the selection of the right partner to the formation of synapses at the right subcellular compartment
What does NGL02 OE do?
Promotes dendritic spine protrusions
Utrophin
Protein that connects dystroglycan and actin. Required for membrane maintenance and clustering of AChRs.
One is protected and one blocked:
Punishment of unprotected, blocked synapse. Induces destabilization and refraction of presynaptic terminal
Experimental Design to show glia astrocytes increase the number of puncta containing both pre/postsynaptic proteins
RGCs cultured in the presence and absence of glia and stained for synaptotagmin and PSD-95
EphBs type of receptors
Receptor tyrosine kinases
What happens in a KO of NGL-2 in dissociated HC neurons?
Reduces the density of excitatory but not inhibitory postsynaptic specializations
4th Mechanism of synapse assembly
Release positive agrin and negative ACh signals from the nerve
Acetylcholine
Represses AChR subunit gene expression in extrasynaptic area
Loss and gain of functino analyses of rapsyn
Required for AChRs clustering
What happens with KO of EphB2 in cortical neurons?
Results in decreased presynaptic specialization; decreased in spines and functional excitatory synaptic inputs
Development of synaptic defects in E17 - Chat -/-
See more clusters of receptors because they aren't dispersed.
What happens in selective genetic disruption of z+ agrin?
Significant defect in NMJ formation.
Dystrophin protein complex
Skeletal formation that makes complex between terminal and synapse. Associated with muscular dystrophy
3rd Mechanism of synapse assembly
Stability of AChR turnover rate (mature vs embryonic AChR)
What to synaptic adhesion molecules do during synaptogenesis?
Stabilize the initial contact between axons and dendrites
How to track synapse maturation?
Sternomastoid muscle was labeled with BTX-Alexa 488 and superficial synapses were imaged at different stages of development.
What are diffusable factors important for and what can they do?
Synapse specificity, 1) guide axonal projections to proper targets 2) induce axonal differentiation and growth 3) promote a favorable environment for synapse formation
What happens after active zone and receptor clusters?
Synapse structure/function matures and synaptic folds, basal lamina form and AChR subunits
2nd Mechanism of synapse assembly
Synapse-specific AChR expression by the nuclei beneath the postsynaptic membrane
When does synapse elimination occur?
The first 2 weeks after birth in mice
3rd category of molecules that control the building of a synapse
Those that modulate synaptic structure and function
1st category of molecules that control the building of a synapse
Those that promote stability of the synapse by linking pre and postsynaptic partners
2nd category of molecules that control the building of a synapse
Those that regulate differentiation of pre and postsynaptic specializations
Dystroglycan
Transmembrane linkage between ECM and the cytoskeleton, serves muscular integrity, and may serve as an agrin receptor and regulate agrin induced AChR clustering
Results of WNT-3 Inducing Axonal Remodeling in Sensory Neurons
WNT-3 induces significant increase in growth cone size and axonal branch orders
Examples of transcription factors used in cell fate signaling
Wnts, BMPs, FGFs
Can growth cone release NT without synapse formation?
Yes, but become a much better at it after target contact
Is synapse elimination activity dependent and what does this mean?
Yes, it only occurs when AP are firing.
1st Possible role of neuregulin in postsynaptic differentiation
act in parallel with agrin; neuregulin might induce acetylcholine receptor (AChR) transcription and agrin might direct AChR clustering
Activity independent processes
differentiation, migration, axon guidance
2nd Possibly role of neuregulin in postsynaptic differentiation
signaling from the axon is essential for Schwann cell survival, and Schwann cells are essential for axonal maintenance. In the absence of Schwann cells, axons might be unable to present agrin adequately
