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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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Neuronal but not muscle agrin activates nsk2/musk promoter fragments, depending on an N-box in the first intron. (A) Expression of luciferase activity under the control of the (1.6-N) promoter fragment in adult soleus muscle fibers in vivo is promoted by neuronal but not my muscle Agrin and is blocked upon mutation of the N-box. Muscle fibers were injected intracellularly with the expression plasmids indicated and with pRL-TK for normalization. (B) Agrin-induced expression of luciferase under the control of various nsk2/musk promoter fragments depends on the presence of the N-box in C2C12 myotubes. C2C12 myoblasts were transfected with pRL-TK and the plasmids indicated and grown and differentiated on a laminin substrate with or without mini-Agrin N257c21B8 attached. (C) Agrin-induced activation of a 5.4-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by removal of the intronic sequence containing the N-box. ***P < 0.001 in two-tailed t test. Bars ± SEM.
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fig3: Neuronal but not muscle agrin activates nsk2/musk promoter fragments, depending on an N-box in the first intron. (A) Expression of luciferase activity under the control of the (1.6-N) promoter fragment in adult soleus muscle fibers in vivo is promoted by neuronal but not my muscle Agrin and is blocked upon mutation of the N-box. Muscle fibers were injected intracellularly with the expression plasmids indicated and with pRL-TK for normalization. (B) Agrin-induced expression of luciferase under the control of various nsk2/musk promoter fragments depends on the presence of the N-box in C2C12 myotubes. C2C12 myoblasts were transfected with pRL-TK and the plasmids indicated and grown and differentiated on a laminin substrate with or without mini-Agrin N257c21B8 attached. (C) Agrin-induced activation of a 5.4-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by removal of the intronic sequence containing the N-box. ***P < 0.001 in two-tailed t test. Bars ± SEM.

Mentions: Expression of endogenous musk can be induced by Agrin/MuSK (Moore et al., 2001). Therefore, we next tested whether the 2.1-kb nsk2/musk promoter fragment conferring synaptic expression could be activated specifically by neuronal Agrin. For this purpose, we injected expression plasmids coding for full-length chicken neuronal Agrin, pcAgrin748, p1.6luc-N, and pRL-TK as a reference along with pnlsGFP-S6 intracellularly into extrasynaptic regions of innervated soleus muscles. As a control, pcAgrin748 was replaced with an expression vector for the nonneuronal isoform of Agrin, pcAgrin700 (Ruegg et al., 1992). 2 wk later, in fiber regions containing GFP-positive nuclei, luciferase activity measured in fibers expressing neuronal Agrin was ∼3.5 times higher than in fibers expressing muscle Agrin (Fig. 3 A). On a “per nucleus” basis, this is a lower estimate because the fiber segments dissected (2–3 mm in length) contained many more GFP-positive nuclei outside the Agrin-induced ectopic, postsynaptic membranes (and thus not activated by Agrin) than nuclei colocalized with such sites. Thus, neuronal Agrin but not muscle Agrin activates the same 2.1-kb nsk2/musk promoter fragment, (1.6 N) as does the nerve.


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)

Neuronal but not muscle agrin activates nsk2/musk promoter fragments, depending on an N-box in the first intron. (A) Expression of luciferase activity under the control of the (1.6-N) promoter fragment in adult soleus muscle fibers in vivo is promoted by neuronal but not my muscle Agrin and is blocked upon mutation of the N-box. Muscle fibers were injected intracellularly with the expression plasmids indicated and with pRL-TK for normalization. (B) Agrin-induced expression of luciferase under the control of various nsk2/musk promoter fragments depends on the presence of the N-box in C2C12 myotubes. C2C12 myoblasts were transfected with pRL-TK and the plasmids indicated and grown and differentiated on a laminin substrate with or without mini-Agrin N257c21B8 attached. (C) Agrin-induced activation of a 5.4-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by removal of the intronic sequence containing the N-box. ***P < 0.001 in two-tailed t test. Bars ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199368&req=5

fig3: Neuronal but not muscle agrin activates nsk2/musk promoter fragments, depending on an N-box in the first intron. (A) Expression of luciferase activity under the control of the (1.6-N) promoter fragment in adult soleus muscle fibers in vivo is promoted by neuronal but not my muscle Agrin and is blocked upon mutation of the N-box. Muscle fibers were injected intracellularly with the expression plasmids indicated and with pRL-TK for normalization. (B) Agrin-induced expression of luciferase under the control of various nsk2/musk promoter fragments depends on the presence of the N-box in C2C12 myotubes. C2C12 myoblasts were transfected with pRL-TK and the plasmids indicated and grown and differentiated on a laminin substrate with or without mini-Agrin N257c21B8 attached. (C) Agrin-induced activation of a 5.4-kb nsk2/musk promoter fragment in C2C12 myotubes is blocked by removal of the intronic sequence containing the N-box. ***P < 0.001 in two-tailed t test. Bars ± SEM.
Mentions: Expression of endogenous musk can be induced by Agrin/MuSK (Moore et al., 2001). Therefore, we next tested whether the 2.1-kb nsk2/musk promoter fragment conferring synaptic expression could be activated specifically by neuronal Agrin. For this purpose, we injected expression plasmids coding for full-length chicken neuronal Agrin, pcAgrin748, p1.6luc-N, and pRL-TK as a reference along with pnlsGFP-S6 intracellularly into extrasynaptic regions of innervated soleus muscles. As a control, pcAgrin748 was replaced with an expression vector for the nonneuronal isoform of Agrin, pcAgrin700 (Ruegg et al., 1992). 2 wk later, in fiber regions containing GFP-positive nuclei, luciferase activity measured in fibers expressing neuronal Agrin was ∼3.5 times higher than in fibers expressing muscle Agrin (Fig. 3 A). On a “per nucleus” basis, this is a lower estimate because the fiber segments dissected (2–3 mm in length) contained many more GFP-positive nuclei outside the Agrin-induced ectopic, postsynaptic membranes (and thus not activated by Agrin) than nuclei colocalized with such sites. Thus, neuronal Agrin but not muscle Agrin activates the same 2.1-kb nsk2/musk promoter fragment, (1.6 N) as does the nerve.

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