Calcium/Calmodulin Kinase II-Dependent Acetylcholine Receptor Cycling at the Mammalian Neuromuscular JunctionIn Vivo

Abstract

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca 2ϩ /calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKII␤m isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKII␥ and ␦ isoforms are poorly expressed in synaptic sites. These results indicate that Ca 2ϩ along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

“…Comparison of recycled AChR pools in normal vs. chronically denervated muscles showed that in the absence of muscle stimulation most internalized receptors are targeted for degradation, and fewer receptors are able to recycle back into denervated NMJs (Bruneau and Akaaboune, 2006). Conversely, stimulation of denervated muscles increased AChR recycling (Martinez-Pena y Valenzuela et al, 2010), suggesting that stimulation-induced AChR re-stabilization in denervated muscle could be explained by increased AChR recycling. Therefore, we asked whether agrin could also increase AChR recycling at agrin-induced AChR clusters in denervated muscle.…”

“…However, as shown previously for synaptic AChRs, full stabilization of AChRs in ectopic, agrin-induced AChR clusters to a half-life of 10–14 days also requires muscle activity. Of note, muscle stimulation can promote the translocation of internalized receptors from the internal pool into the postsynaptic membrane in denervated synapses (Martinez-Pena y Valenzuela et al, 2010). Thus, the increase in AChR stability by muscle activity could be explained at least in part by an increase in the delivery of recycled AChR into induced clusters.…”

Impact of Sustained Exposure to β-Amyloid on Calcium Homeostasis and Neuronal Integrity in Model Nerve Cell System Expressing α4β2 Nicotinic Acetylcholine Receptors

“…Our results for endocytotic and recycling markers indicate that an up-regulation of this pathway did occur, which was confirmed in a functional assay of nAChR recycling, suggesting that an increase in endocytosis/ receptor recycling may account, in part, for the observed receptor up-regulation upon prolonged exposure to A␤. How this up-regulation in recycling is induced will be the subject of future studies, focusing, in particular, on calcium-regulated pathways (52) and A␤ internalization (53). Nonetheless, it is proposed that up-regulation of the nAChRs with sustained, elevated levels of A␤ leads to a feed-forward dysregulation of calcium homeostasis.…”

Initiation of synapse formation by Wnt-induced MuSK endocytosis

“…4). Although we cannot exclude the possibility that rab11 exerts its function directly on AChR trafficking, recycling of AChR receptors has been observed only at mature NMJs and only in response to muscle stimulation (Bruneau and Akaaboune, 2006;Martinez-Pena y Valenzuela et al, 2010), whereas at the stage of AChR pre-patterning when we examined rab11-dependent trafficking, NMJs have not yet formed. Combined with the observations that MuSK is crucial for localization of Dishevelled to presumptive endosomes, and that Dishevelled function in muscle cells is crucial for AChR pre-patterning and axon guidance (see below) (Jing et al, 2009), we favor the idea that rab11 regulates primarily MuSK localization and function.…”