Synapse Formation I April 18, 2007 Mu-ming Poo 1.Steps in the formation of neuromuscular junction 2.Early events of synaptogenesis 3.ACh receptor aggregation at the postsynaptic membrane ---- The agrin hypothesis ---- The agrin-ACh hypothesis 4.Activity-dependent synapse maturation

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

Steps in the development of the neuromuscular junction (NMJ) 1.Presynaptic motor axon express ACh 2.ACh release from growth cones detected 3.Growth cone contact of the myotube surface 4.Evoked ACh detected in the muscle cell 5.Pre- and postsynaptic differentiation -- formation presynaptic active zones -- postsynaptic ACh receptor clusters. 6.Maturation of synapse structure & function -- formation of synaptic folds, basal lamina -- switching of ACh receptor subunits 7.Activity-dependent competition – elimination of polyneuronal innervation

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)

Accumulation of a variety of synaptic components at NMJs

Mechanisms for the formation of postsynaptic AChR aggregates 1.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!!

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

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 Search for AChR aggregation factors AChR Clusters induced by agrin without nerve in cultured myotubes

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,

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.

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)

Three processes leading to postsynaptic AChR aggregates 1.Redistribution of surface AChRs (clustering). 2.Local increase of ACh synthesis (by synaptic nuclei) 3.Global down-regulation of AChR synthesis (by extrasynaptic nuclei)

The Agrin Hypothesis The textbook’s view:

Problems with the agrin hypothesis of AChR clustering 1.Several aneural KO mice (with erbB, topoisomerase deleted) showed formation of AChRs in the central endplate band of the aneural muscle 2.Agrin KO mice showed transient AChR clusters before birth ---nerve stablizes the clusters? 3.Nuclei associated with AChR clusters in aneural muscle became transcriptionally specialized 4.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.”

The role of ACh 1.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 1.In Agrin KO, AChR clusters still form but not stable 2.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.

Kummer, et al. “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. The Current View: