Limits...
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.

Show MeSH

Related in: MedlinePlus

Agrin activates nsk2/musk via an ErbB- independent pathway in muscle fibers in vivo. (A) Activation of the nsk2/musk promoter fragment by Agrin in vivo is marginally affected by HER2KM and HER4KM but is fully blocked by JNK-APF. MKK7D activates the nsk2/musk fragment in the absence of Agrin. Nonspecific background luciferase activities as observed in muscle fibers injected with expression vector for muscle agrin (pcAgrin700; see Fig. 3 A) are subtracted. (B) Injection of pHERKM induces expression of HERKM in muscle fibers in vivo as myc immunoreactivity was observed at agrin-induced ectopic postsynaptic membranes upon injecting pmyc_HER2KM, a fusion of HER2KM and myc. Bar, 25 μm. (C) JNK-APF inhibits ectopic subsynaptic differentiation by Agrin. Muscle fibers were injected with pcAgrin748, pnslGFP, and pJNK-APF or equivalent amounts of empty vector (control); the formation of ectopic AChR clusters in GFP-positive fibers was then examined in cross sections, and injected fibers were identified by their expression of GFP. (Top) Control fiber (note the formation of AChR cluster [red] in GFP-positive control fiber [green nuclei]). (Middle) Agrin secreted induces AChR cluster on adjacent, GFP-negative (JNK-APF–negative) but not on GFP- (JNK-APF) expressing fiber. Bar, 25 μm. (Bottom) The percentage of GFP-positive fibers forming AChR clusters in response to Agrin is depressed in JNK-APF compared with control fibers. Each data point represents one muscle. 83–513 fiber profiles were examined per muscle.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199368&req=5

fig6: Agrin activates nsk2/musk via an ErbB- independent pathway in muscle fibers in vivo. (A) Activation of the nsk2/musk promoter fragment by Agrin in vivo is marginally affected by HER2KM and HER4KM but is fully blocked by JNK-APF. MKK7D activates the nsk2/musk fragment in the absence of Agrin. Nonspecific background luciferase activities as observed in muscle fibers injected with expression vector for muscle agrin (pcAgrin700; see Fig. 3 A) are subtracted. (B) Injection of pHERKM induces expression of HERKM in muscle fibers in vivo as myc immunoreactivity was observed at agrin-induced ectopic postsynaptic membranes upon injecting pmyc_HER2KM, a fusion of HER2KM and myc. Bar, 25 μm. (C) JNK-APF inhibits ectopic subsynaptic differentiation by Agrin. Muscle fibers were injected with pcAgrin748, pnslGFP, and pJNK-APF or equivalent amounts of empty vector (control); the formation of ectopic AChR clusters in GFP-positive fibers was then examined in cross sections, and injected fibers were identified by their expression of GFP. (Top) Control fiber (note the formation of AChR cluster [red] in GFP-positive control fiber [green nuclei]). (Middle) Agrin secreted induces AChR cluster on adjacent, GFP-negative (JNK-APF–negative) but not on GFP- (JNK-APF) expressing fiber. Bar, 25 μm. (Bottom) The percentage of GFP-positive fibers forming AChR clusters in response to Agrin is depressed in JNK-APF compared with control fibers. Each data point represents one muscle. 83–513 fiber profiles were examined per muscle.

Mentions: Next, we examined whether these pathways could be resolved in muscle fibers in vivo. Unlike in C2C12 cells, activation of nsk2/musk by Agrin was not blocked in adult fibers by overexpression of the inactive ErbB mutants. Specifically, upon injection with pHER2KM and pHER4KM into muscle fibers in vivo, activation of p1.6luc-N by neuronal Agrin was not significantly reduced compared with fibers injected with wild-type pHER2 and pHER4 (Fig. 6 A). Expression of pHERKM and accumulation of HER2KM at ectopic sites was ascertained by immunocytochemistry (Fig. 6 B). Similar results were obtained with the AChRɛ promoter–reporter construct (unpublished data).


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)

Agrin activates nsk2/musk via an ErbB- independent pathway in muscle fibers in vivo. (A) Activation of the nsk2/musk promoter fragment by Agrin in vivo is marginally affected by HER2KM and HER4KM but is fully blocked by JNK-APF. MKK7D activates the nsk2/musk fragment in the absence of Agrin. Nonspecific background luciferase activities as observed in muscle fibers injected with expression vector for muscle agrin (pcAgrin700; see Fig. 3 A) are subtracted. (B) Injection of pHERKM induces expression of HERKM in muscle fibers in vivo as myc immunoreactivity was observed at agrin-induced ectopic postsynaptic membranes upon injecting pmyc_HER2KM, a fusion of HER2KM and myc. Bar, 25 μm. (C) JNK-APF inhibits ectopic subsynaptic differentiation by Agrin. Muscle fibers were injected with pcAgrin748, pnslGFP, and pJNK-APF or equivalent amounts of empty vector (control); the formation of ectopic AChR clusters in GFP-positive fibers was then examined in cross sections, and injected fibers were identified by their expression of GFP. (Top) Control fiber (note the formation of AChR cluster [red] in GFP-positive control fiber [green nuclei]). (Middle) Agrin secreted induces AChR cluster on adjacent, GFP-negative (JNK-APF–negative) but not on GFP- (JNK-APF) expressing fiber. Bar, 25 μm. (Bottom) The percentage of GFP-positive fibers forming AChR clusters in response to Agrin is depressed in JNK-APF compared with control fibers. Each data point represents one muscle. 83–513 fiber profiles were examined per muscle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199368&req=5

fig6: Agrin activates nsk2/musk via an ErbB- independent pathway in muscle fibers in vivo. (A) Activation of the nsk2/musk promoter fragment by Agrin in vivo is marginally affected by HER2KM and HER4KM but is fully blocked by JNK-APF. MKK7D activates the nsk2/musk fragment in the absence of Agrin. Nonspecific background luciferase activities as observed in muscle fibers injected with expression vector for muscle agrin (pcAgrin700; see Fig. 3 A) are subtracted. (B) Injection of pHERKM induces expression of HERKM in muscle fibers in vivo as myc immunoreactivity was observed at agrin-induced ectopic postsynaptic membranes upon injecting pmyc_HER2KM, a fusion of HER2KM and myc. Bar, 25 μm. (C) JNK-APF inhibits ectopic subsynaptic differentiation by Agrin. Muscle fibers were injected with pcAgrin748, pnslGFP, and pJNK-APF or equivalent amounts of empty vector (control); the formation of ectopic AChR clusters in GFP-positive fibers was then examined in cross sections, and injected fibers were identified by their expression of GFP. (Top) Control fiber (note the formation of AChR cluster [red] in GFP-positive control fiber [green nuclei]). (Middle) Agrin secreted induces AChR cluster on adjacent, GFP-negative (JNK-APF–negative) but not on GFP- (JNK-APF) expressing fiber. Bar, 25 μm. (Bottom) The percentage of GFP-positive fibers forming AChR clusters in response to Agrin is depressed in JNK-APF compared with control fibers. Each data point represents one muscle. 83–513 fiber profiles were examined per muscle.
Mentions: Next, we examined whether these pathways could be resolved in muscle fibers in vivo. Unlike in C2C12 cells, activation of nsk2/musk by Agrin was not blocked in adult fibers by overexpression of the inactive ErbB mutants. Specifically, upon injection with pHER2KM and pHER4KM into muscle fibers in vivo, activation of p1.6luc-N by neuronal Agrin was not significantly reduced compared with fibers injected with wild-type pHER2 and pHER4 (Fig. 6 A). Expression of pHERKM and accumulation of HER2KM at ectopic sites was ascertained by immunocytochemistry (Fig. 6 B). Similar results were obtained with the AChRɛ promoter–reporter construct (unpublished data).

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