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A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

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Model for stabilization of synaptic gene expression through stabilization of musk expression by agrin from motor nerve terminal. Agrin secreted from nerve terminal activates preexisting MuSK to induce expression of musk via its N-box (i) by organizing an NRG/ErbB pathway, involving MuSK-induced recruitment of ErbB receptors and of muscle-derived NRG and (ii) by MuSK-induced activation of JNK (via Rac/Cdc42). With musk expression stabilized, the same pathways are used for AChR and erbB expression. Expression may be strengthened by NRG-1 secreted from nerve terminal. Complete inhibition of Agrin-induced musk transcription in C2C12 cells by overexpression of inactive ErbBs (HER2KM and HER4KM) and by dominant-negative JNK suggests that the two pathways are connected. The model is based on present data (solid arrows) and references cited (broken arrows).
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fig7: Model for stabilization of synaptic gene expression through stabilization of musk expression by agrin from motor nerve terminal. Agrin secreted from nerve terminal activates preexisting MuSK to induce expression of musk via its N-box (i) by organizing an NRG/ErbB pathway, involving MuSK-induced recruitment of ErbB receptors and of muscle-derived NRG and (ii) by MuSK-induced activation of JNK (via Rac/Cdc42). With musk expression stabilized, the same pathways are used for AChR and erbB expression. Expression may be strengthened by NRG-1 secreted from nerve terminal. Complete inhibition of Agrin-induced musk transcription in C2C12 cells by overexpression of inactive ErbBs (HER2KM and HER4KM) and by dominant-negative JNK suggests that the two pathways are connected. The model is based on present data (solid arrows) and references cited (broken arrows).

Mentions: Next, we asked whether Agrin could also activate musk via a direct shunt from MuSK. For example, an alternative way for Agrin to activate JNK in C2C12 cells is via the Rho family GTPases Rac and Cdc42 (Weston et al., 2000; Luo et al., 2002). Consistent with such a mechanism, constitutively active Rac, RacV12, strongly activated p(1.6luc-N) in the absence of Agrin. This effect was abolished by GABPβDN (Fig. 5 D), suggesting that Rac activates nsk2/musk via the N-box only. If Agrin-induced activation of musk via Rac occurs independently of NRG/ErbB, Agrin will induce p1.6luc-N more strongly than saturating concentrations of NRG-1, and induction will be blocked by a dominant-negative mutant of Rac, RacN17. In normal C2C12 myotubes, no difference in the activation of p1.6luc-N by Agrin and by NRG-1 was resolved (unpublished data). However, when ErbB signaling was blocked by overexpression of inactive ErbB mutants, Agrin activated p(1.6luc-N) more strongly than saturating concentrations of NRG-1 (2 nM), which was not due to increased levels of ErbB2 (Fig. 5 F). Furthermore, activation was completely blocked by RacN17 (Fig. 5 E). Together, these data suggest that Agrin/MuSK can activate nsk2/musk via two pathways (Fig. 7): (a) via a direct shunt activated by MuSK involving Rac and (b) via the organization of a secondary NRG-1/ErbB pathway. Both pathways converge onto GABP. In C2C12 myotubes, the secondary NRG-1/ErbB pathway occludes the MuSK-induced shunt.


A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Model for stabilization of synaptic gene expression through stabilization of musk expression by agrin from motor nerve terminal. Agrin secreted from nerve terminal activates preexisting MuSK to induce expression of musk via its N-box (i) by organizing an NRG/ErbB pathway, involving MuSK-induced recruitment of ErbB receptors and of muscle-derived NRG and (ii) by MuSK-induced activation of JNK (via Rac/Cdc42). With musk expression stabilized, the same pathways are used for AChR and erbB expression. Expression may be strengthened by NRG-1 secreted from nerve terminal. Complete inhibition of Agrin-induced musk transcription in C2C12 cells by overexpression of inactive ErbBs (HER2KM and HER4KM) and by dominant-negative JNK suggests that the two pathways are connected. The model is based on present data (solid arrows) and references cited (broken arrows).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199368&req=5

fig7: Model for stabilization of synaptic gene expression through stabilization of musk expression by agrin from motor nerve terminal. Agrin secreted from nerve terminal activates preexisting MuSK to induce expression of musk via its N-box (i) by organizing an NRG/ErbB pathway, involving MuSK-induced recruitment of ErbB receptors and of muscle-derived NRG and (ii) by MuSK-induced activation of JNK (via Rac/Cdc42). With musk expression stabilized, the same pathways are used for AChR and erbB expression. Expression may be strengthened by NRG-1 secreted from nerve terminal. Complete inhibition of Agrin-induced musk transcription in C2C12 cells by overexpression of inactive ErbBs (HER2KM and HER4KM) and by dominant-negative JNK suggests that the two pathways are connected. The model is based on present data (solid arrows) and references cited (broken arrows).
Mentions: Next, we asked whether Agrin could also activate musk via a direct shunt from MuSK. For example, an alternative way for Agrin to activate JNK in C2C12 cells is via the Rho family GTPases Rac and Cdc42 (Weston et al., 2000; Luo et al., 2002). Consistent with such a mechanism, constitutively active Rac, RacV12, strongly activated p(1.6luc-N) in the absence of Agrin. This effect was abolished by GABPβDN (Fig. 5 D), suggesting that Rac activates nsk2/musk via the N-box only. If Agrin-induced activation of musk via Rac occurs independently of NRG/ErbB, Agrin will induce p1.6luc-N more strongly than saturating concentrations of NRG-1, and induction will be blocked by a dominant-negative mutant of Rac, RacN17. In normal C2C12 myotubes, no difference in the activation of p1.6luc-N by Agrin and by NRG-1 was resolved (unpublished data). However, when ErbB signaling was blocked by overexpression of inactive ErbB mutants, Agrin activated p(1.6luc-N) more strongly than saturating concentrations of NRG-1 (2 nM), which was not due to increased levels of ErbB2 (Fig. 5 F). Furthermore, activation was completely blocked by RacN17 (Fig. 5 E). Together, these data suggest that Agrin/MuSK can activate nsk2/musk via two pathways (Fig. 7): (a) via a direct shunt activated by MuSK involving Rac and (b) via the organization of a secondary NRG-1/ErbB pathway. Both pathways converge onto GABP. In C2C12 myotubes, the secondary NRG-1/ErbB pathway occludes the MuSK-induced shunt.

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Show MeSH
Related in: MedlinePlus