Lecture 13

Lecture 13

1. What is a model system for study of synapse components and formation?

   • Neuromuscular Junction (NMJ)
     • What are the morphological features of a mature NMJ?
       • 1. Pre and postsynaptic membranes are separated by synaptic cleft (has basal lamina) and extracellular matrix components
         • What is the basal lamina?
           • It is the organized sheets of extracellular matrix components (Connective tissue)
       • 2. Vesicles are clustered at presyn release sites (active zones), transmitter receptors are clustered in the postsynaptic membrane
         • What is postsynaptic density PSD?
           • It is the clustering of AChRs at the junctional folds
       • 3. Nerve terminals are coated by Schwann cell processes.
     • In which direction are the signaling events of the NMJ?
       • They are defined as bidirectional signaling events
         • Ex) pre: MN talks to post muscle cell : secrete signaling molecules to tell the cell to organize and get ready
           • post--> pre muscle cell can tell the MN to organize the active zone to the presynaptic areas
     • How many stages does the NMJ develop in?
       • 5
         • Number One: The growth cone approaches:
           • Prior to contact, MN releases ACh, and AChR expressed on the muscle membrane can detect them
         • Number Two: MN contact with muscle
           • AChRs on the muscle show dramatic clustering and MN forms vesicle rich terminal arborizations
         • Number 3: Basal lamina starts to form
           • Upregulation of synaptic nAChRs Downregulation of extrasynaptic /extrajunctional nAChRs
         • Number Four: Multiple axons converge on the same muscle fiber
           • (innervated by multiple neurons) initially
         • Number Five: Synaptic Pruning
           • Only one axon per muscle in mature NMJ, monosynaptic
     • What are the four signaling events of the NMJ in summary?
       • 1) Post--> Pre : Laminin-diff of active zone
         • What is laminin?
           • It is an extracellular matrix molecule (ECM) and major component of basal lamina
             • What does laminin promote?
               • Axon outgrowth in many neuronal types due to diff of active zones
         • Where are laminins synthesized then kept?
           • Synthesized by muscle cells (post) and incorporated into the basal lamina
             • What is the mechanism of the laminins promoting the diff of the active sites?
               • They bind to VGCCs on the axon terminal, leading to the recruitment of other components of the release apparatus ; accumulate SVs, stop growing, and acquire ability to release NT
         • Experiments with laminin
           • Experiment One
             • The denervation (cut off axon) experiment: for the neuron to regrow, the NMJ junction forms at the same site at the old one
               • Conclusion: something from the muscle is determining the location of the NMJ (where to form the synapse specifically)
           • Experiment Two
             • In order to determine if the laminin is presented on the muscle cells or it's something secreted, they removed the muscle and left the basal lamella Result: The NMJ still forms at the same site
               • Conclusion: Lamins are synthesized by muscle cells and incorporated into the basal lamina
       • 2) Pre--> Post Agrin--> promote clustering of AChRs on muscle
         • What is Agrin?
           • A large multi-domain proteoglycan
           • What does Agrin do?
             • Released from pre-syn MN terminals to cause postsyn AChR clustering
           • Where is Agrin Secreted?
             • It's secreted by MN into basal lamina
             • What does agrin signaling depend on?
               • MuSK (muscle specific trk-related receptor w/ kringle domain) (Receptor Tyrosine Kinase= expressed on muscle surface)
                 • What interaction protein does MuSK activate?
                   • The activated MuSK recruits Rapsyn, an AChR interacting protein
                     • How does rapsyn interact with AChR to induce clustering?
                       • 1) upregulates synaptic AChR synthesis 2) Represses extrasynaptic AChR synthesis
               • What is observed in agrin/MuSK knockouts?
                 • There are no observed clustering of AChRs
       • 3) Pre--> Post: neuregulin--> promote transcription AChR at junctional nuclei
         • What does neuregulin do?
           • It is released by the pre-syn MN to promote AChR synthesis (not clustering)
             • AChR transcription is enhanced in junctional nuclei
               • Also acts on Schwann cells to promote their contribution
         • What is neuregulin exactly?
           • ARIA = acetylcholine receptor inducing activity, a trophic factor
         • What is the mechanism of neuregulin?
           • ARIA activates erbB receptor tyrosine kinase
             • Activates the small g-protein ras
               • activates MAPK
                 • Activates the transcription of AChRs
         • What do neuregulin heterozygotes show?
           • 50% reduction in AChR density Reduced amplitude of MEPP (muscular Excitatory post synaptic potential )
       • 4) ACh as a dispersal factor a. downregulate ACh expression at extrajunctional site b. internalization of AChR at extrajunctional sites
         • How does ACh itself, a NT, function as a signaling molecule of NMJs?
           • It is released by the pre-syn MN to suppress extrajunctional AChR synthesis
         • What is the mechanism of ACh inhibiting extrajunctional AChR synthesis?
           • Muscle Depolarization by ACh, leads to Ca2+ influx
             • kinase activation
               • phosphorylation of transcription factors
                 • inactivation --> shut off AChR transcription globally
         • What is observed when there is denervation or paralysis?
           • Upregulation of AChR synthesis ( activity needed for repression)
           • What does direct electrical stimulation of muscle also prove?
             • That activity is required for repressing AChR transcription and promotion of AChR internalization
         • ACh, disperses nAChR clusters !
           • How does agrin may render AChRs immune to the de-clustering effects of ACh?
             • Agrin represses nAChR loss locally (at nerve contact) to promote synapse formation.

2. PNS pruning

   • Elimination of multiple innervations in the PNS
     • P11- two axons innervate same muscle
     • P12- green axon terminal expands
     • P14- blue axon fully retracted, synaptic terminal transformed into large retraction bulb, then fully withdrawn from synaptic site
     • Two weeks after birth each fiber is innervated by only a single MN, Why?
       • To ensure that each muscle fiber is innervated, allow axons to catch appropriate number of target cells
   • Blocking AChR function leads to what pruning mechanism?
     • It locally leads to the retraction of nerve terminals (activity-dependent)
   • Synaptic elimination is a process that is regulated by muscle and neuronal activity:
     • local blockade of AChR - injection of curare

3. CNS development

   • Where does the excitatory central synapse tend to form at?
     • At the Dendritic spine
   • Where does the inhibitory central synapse form?
     • At the Dendritic shaft
       • What are located on dendritic shafts where microtubules are abundant ?
         • most GABAnergic synapses
   • What can be observed in both cases of +/- central synapses?
     • Active zone: presynaptic region where SV fusion can occur (visible in EM meshwork of proteins) Postsynaptic density: oppositing active zone, clusters of NT Receptors, channels, signaling molecules, and scaffolding proteins

4. Synaptogenesis

   • What is the first step of synaptogenesis?
     • Initial contact: cell adhesion molecules (CAMs)
       • cadherins/protocadherins work as adhesive factors of the pre and postsynaptic membranes
         • We can encode a large number of cadherin subtypes
           • leading to variable extracellular domains, and constant intracellular domain.
         • What type of interaction does cadherin use?
           • homophilic interaction; creates synaptic zipcodes
   • What is the second step of synaptogenesis?
     • Presynaptic induction: additional CAMs (inductive factors)
       • induce the formation of presynaptic active zones and stabilize the nascent synaptic junction
         • What homophillic interaction of Cadherins do: also trigger the accumulation of Presynaptic differentiation of the active zone, and upregulation of SVs
       • What are four examples of inductive factors ( additional CAMs)
         • SynCAM
         • Nxn/NL
           • What are heterophillic interactions of inductive factors?
             • neurexin (pre) + neuroligin (post);
               • interacts with synaptotacin (ca sensor)
                 • localizes VGCC on pre-syn side, trigger differentiation of the active zone
                   • maturation of NMDA receptor Align pre-post synaptic components
             • Neuroligin interacts with PSD-95
               • PSD 95: (post syn scaffolding protein for Glu synapses)
                 • 1. clustering of NT receptors 2. formation of PSD 3. aligns the two components of pre and post
         • Narp
         • EphrinB/EphBR
           • Ephrin ligands / Eph receptor tyrosin kinase:
             • Both receptors initiate intracellular signaling events (Eph REVERSES)
               • EphrinB (pre) + EphB receptor (post) : activation leads to
                 • EphB receptor aggregation, important for clustering of NMDA-Rs
                   • (calcium permeability goes up ) also helps dendritic spine development