. Neurotransmitter Acetylcholine Negatively Regulates Neuromuscular Synapse Formation by a Cdk5-Dependent Mechanism. Neuron. 2005 May 1;46:569.

Abstract:

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

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  1. Benjamin A. Samuels and Li-Huei Tsai Postsynaptic clusters of acetylcholine receptors (AChR) form at the developing neuromuscular junction (NMJ) prior to apposition by the presynaptic nerve terminal. Subsequently, clusters that are innervated by the nerve terminal become stable, while clusters that are not innervated disperse. It is believed that an important positive signal for stabilization is agrin, but until recently, a signal for dispersion had not been discovered. A recent paper from the lab of Kuo-Fen Lee provides evidence that the Cdk5 kinase plays an essential role in this process (1).

    Cdk5 is a small serine/threonine kinase that is primarily active in neurons. However, a report from the lab of Nancy Ip in 2001 first established a potentially important role for Cdk5 in the postsynaptic myotubes of the NMJ by showing it promoted neuregulin-induced transcription of the AChR (2). An abstract from Nancy Ip’s lab in 2003 also suggested that agrin-induced AChR clusters are significantly larger in myotubes cultured from Cdk5-deficient mice (3). Similarly, the manuscript from the Lee lab shows that in Cdk5-deficient mice AChR clusters are larger and more numerous.

    To gain a better understanding of the importance of Cdk5, the authors created an agrin/Cdk5 double knockout mouse and investigated the NMJ. Very few AChR clusters are present in agrin-deficient mice because the clusters cannot be stabilized and therefore become actively dispersed. Remarkably, clusters are numerous in the double knockout mice, demonstrating that Cdk5 is necessary for the dispersion. Similar results were obtained with a Cdk5 inhibitor.

    Interestingly, the authors also found that the AChR agonist carbachol activates Cdk5 in myotubes. Thinking that perhaps acetylcholine is necessary for activating Cdk5 and inducing AChR dispersion in vivo, the authors investigated NMJ development in mice deficient for ChAT, the key enzyme in acetylcholine synthesis. Similar to the Cdk5-deficient mice, ChAT knockouts had larger and more numerous AChR clusters than did wild-type mice. Also, agrin/ChAT double knockouts had numerous AChR clusters when very few were found in agrin-deficient mice. Further experiments in cultured myotubes using Cdk5 inhibitors in conjunction with AChR agonists lend credibility to a model whereby the neurotransmitter acetylcholine is released, activates Cdk5 in myotubes, and results in dispersion of AChR clusters from sites that are not going to be innervated.

    While many of the specifics remain to be worked out at the NMJ, this intriguing model for Cdk5 regulating postsynaptic structures is not without precedent. In neurons of Cdk5-deficient mice, PSD-95 clusters are larger and more numerous than in wild-type (4), suggesting that Cdk5 activity negatively impacts on NMDA receptor clustering. This idea of Cdk5 activity negatively regulating the size of postsynaptic receptor clusters, and perhaps the postsynaptic density, seems to fit well with the proposed role of Cdk5 at the NMJ in the Lee paper. The study of Cdk5 at the synapse is still in its infancy, but more and more evidence is accumulating that this kinase is a major player. Postsynaptically, Cdk5 also phosphorylates the NMDA receptor (5,6) and participates in a calcium-dependent complex with CaMKII and ╬▒-actinin-1 (7). Cdk5 is not just limited to the postsynaptic side as it also phosphorylates Munc-18 (8,9), Dynamin-1 (10,11), Amphiphysin-1 (12), Synaptojanin-1 (13) and PIPKI╬│ (14), all of which are critical proteins in the synaptic vesicle cycle. Future studies of Cdk5 will clearly focus on its role at the synapse.

    See also:

    Fu AK et al. J Neurochem 87 S1, 168 (2003).

    References:

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    . Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses. Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):546-51. PubMed.

    . Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases. J Cell Biol. 2005 Feb 28;168(5):789-99. PubMed.