Synapse Formation I April 18, 2007 Mu-ming Poo Steps in the formation of neuromuscular junction

1. Synapse Formation I • April 18, 2007 Mu-ming Poo • Steps in the formation of neuromuscular junction • Early events of synaptogenesis • ACh receptor aggregation at the postsynaptic membrane • ---- The agrin hypothesis • ---- The agrin-ACh hypothesis • Activity-dependent synapse maturation

3. Development of the muscle fiber and neuromuscular junction Ann. Rev. Neurosci. (1999)

4. Steps in the development of the neuromuscular junction (NMJ) • Presynaptic motor axon express ACh • ACh release from growth cones detected • Growth cone contact of the myotube surface • Evoked ACh detected in the muscle cell • Pre- and postsynaptic differentiation • -- formation presynaptic active zones • -- postsynaptic ACh receptor clusters. • Maturation of synapse structure & function • -- formation of synaptic folds, basal lamina • -- switching of ACh receptor subunits • Activity-dependent competition • – elimination of polyneuronal innervation

5. Initial events in synaptogenesis • Growth cone of the spinal motor neuron can release ACh before synapse formation • Muscle fibers have ACh receptors (AChRs) before innervation by motor neurons (the AChR density is uniform along the fiber)

6. Accumulation of a variety of synaptic components at NMJs

7. Mechanisms for the formation of postsynaptic AChR aggregates • Redistribution - diffusion of AChRs in the plasma membrane • 2. Local synthesis and insertion of new AChRs at the postsynaptic sites • 3. Reduction of extrajunctional AChRs – electrical activity reduces AChR number All three mechanisms are involved!!

8. 1. Clustering of ACh receptors 1. AChRs are uniformly distributed at high density (100-1000/mm2) in the embryonic myotube membrane. 2. Nerve contact triggers the formation of AChR clusters (10000/mm2), and disappearance of extrasynaptic AChRs (density < 10 /mm2) AChR clustering labeled with a-bungarotoxin (a-Btx), a cobra snake toxin that binds irreversibly with AChR

9. Search for AChR aggregation factors Agrin -- Secreted by motor neuron, muscle and Schwann cell, but neuronal form 1000X more effective -- Promotes AChR clustering without enhancing AChR synthesis -- Neuronal agrin knockout mice showed no clustering Neuregulin (ARIA: AChR-Inducing Activity) -- Secreted by motor neurons -- Promotes AChR subunit synthesis via activation of subsynaptic nuclei, resulting increased surface AChRs AChR Clusters induced by agrin without nerve in cultured myotubes

10. MUSK/rapsin mediate AChR clustering induced by agrin -- Genetic deletion of either MUSK (muscle specific kinase) or rapsin (an anchoring protein) prevent postsynaptic clustering of AChRs -- Expression of AChR in cultured non-muscle cells yielded diffuse distribution of AChRs, but co-expression of AChR and rapsin resulted in AChR clustering,

11. 2. Synthesis of AChR is regulated by neuregulin Neuregulin/ARIA secreted by motor nerve terminal induces an increase in AChR gene transcription in the subsynaptic nuclei.

12. 3. Reduction of extrajunctional AChRs density depends on nerve/muscle activity • Cut nerve or block AChR function results in maintenance of high extrasynaptic AChR density • Stimulate the muscle reduces extrasynaptic AChR density (ACh hypersensitivity) (ACh hypersensitivity)

13. Three processes leading to postsynaptic AChR aggregates • Redistribution of surface AChRs (clustering). • Local increase of ACh synthesis (by synaptic nuclei) • Global down-regulation of AChR synthesis (by extrasynaptic nuclei)

14. The textbook’s view: The Agrin Hypothesis

15. Problems with the agrin hypothesis of AChR clustering • Several aneural KO mice (with erbB, topoisomerase deleted) showed formation of AChRs in the central endplate band of the aneural muscle • Agrin KO mice showed transient AChR clusters before birth • ---nerve stablizes the clusters? • Nuclei associated with AChR clusters in aneural muscle became transcriptionally specialized • Direct in vivo imaging of zebrafish NMJ showed postsynaptic AChR cluster formed prior to axon contact, but preventing axon outgrowth resulted in eventual loss of AChR clusters • “Postsynaptic AChR clusters form prior to innevation, but their maintenance requires innervation.”

16. The role of ACh • ChAT (choline acetyltransferase) KO: ACh in motor axons eliminated, showed abnormal nerve branching , larger postsynaptic AChR clusters – activity restricts cluster growth? • 2. ACh (synaptic activity) down-regulates AChR gene expression. • The role of Agrin • In Agrin KO, AChR clusters still form but not stable • In aneural muscles (no ACh nor agrin) AChR clusters persisted longer than innervated muscle of agrin KO muscle, suggesting agrin counteracts the effect of another nerve-derived factor (ACh?) • 3. Agrin-ChAT double KOs: NMJs formed with normal AChR clusters – inactivity rescued the agrin KO phenotye, agrin is dispensible in the absence of synaptic activity.

17. Kummer, et al. The Current View: “Agrin-ACh Hypothesis” Agrin represses ACh-induced loss of AChRs Agrin acts as anti-declustering factor rather than (or perhaps as well as) clustering factor, and shapes the endplate topography arround the axon arbor.