Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

Abstract

Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ.

Keywords: Frizzled receptors; Wnt proteins; acetylcholine receptor; neuromuscular junction; postsynaptic; skeletal muscle.

Figure 1

Most Fzd receptors are expressed in skeletal muscle cells. Total RNAs were extracted from 6-week old mouse diaphragm and cultured myotubes and further subjected to RT-PCR to detect the mRNA expression of all ten mouse Fzd receptors (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). Fzd receptors 1 to 10 are expressed in the mouse diaphragm (left panel). In cultured C2C12 myotubes, almost all Fzd receptors are expressed at the mRNA level, except for Fzd8 and Fzd10, which were not amplified by RT-PCR (right panel). GAPDH expression was used as a loading control gene. Gels are representative of at least three experiments performed by triplicate.

Figure 2

Fzd9 is dynamically expressed in skeletal muscle tissue. (A) Total RNAs were extracted from E14.5 and E19.5 mouse hind limb muscles and further subjected to RT-PCR to detect the mRNA expression of Fzd9 (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). GAPDH expression was used as a loading control gene. The gel is representative of two experiments performed by triplicate. (B) Proteins from mouse hind limb skeletal muscles obtained at different developmental stages (E14.5, E16.5, and E19.5) were fractionated by SDS-PAGE and immunoblotted for Fzd9 and β-actin (upper panel). Fzd9 is expressed at the protein level in all the analyzed developmental stages since the expected 56kDa band is detected. A representative gel shows that Fzd9 band intensity displays a progressive decrease from E14.5 to E19.5. Data represent the mean ± SD of Fzd9/β-actin ratio from three experiments, normalized to E14.5 (lower panel) (^*p < 0.05, ^**p < 0.01, ANOVA using Bonferroni's post-hoc analysis). (C) Whole-mounted diaphragms from E17.5 and P0 were stained with anti Fzd9 antibody (green) together with αBTX to reveal the postsynaptic densities (blue). Fzd9 is abundant in the synaptic domain of embryonic NMJs, where it displays a punctate expression pattern. The insets (lower panels) show that some αBTX-positive regions were not labeled with anti Fzd9 antibodies. Pictures are representative of at least three experiments performed by triplicate.

Figure 3

Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel). The merge image (lower panel) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions (left panel). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction (right panel). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. (n = 3 performed by triplicate; normalized to GFP-transfected myotubes). (^**p < 0.01, ^***p < 0.001 compared to GFP controls, t-test).

Figure 4

Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate (^**p < 0.01, ^***p < 0.001, t-test). (C) Myoblasts were transfected either with GFP or shFzd9 and differentiated for 5 days. Myotubes were treated with neural agrin and further stained with an anti-Fzd9 antibody (red), along with αBTX (blue) to detect AChR clusters and DAPI (yellow) to stain nuclei. shFz9-transfected myotubes display silenced Fzd9 expression and an apparent increase in the number of AChR clusters compared to GFP-expressing myotubes. (D) Myoblasts transfected either with GFP or shFzd9 and grown for 5 days were treated with neural agrin and further stained with αBTX to detect AChR clusters (red). Myotubes expressing the GFP protein present in the shFzd9 plasmid show a significant increase in the number of AChR clusters, an increase in the total area of AChR clusters, as well as on the average size of AChR clusters, when compared to control myotubes that only express GFP. Data represent the mean ± s.e.m. (n = 3 performed by triplicate; normalized to GFP-transfected myotubes). (^*p < 0.05, ^***p < 0.001 compared to GFP controls, t-test).

Figure 5

Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data (right panel) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes (right panel). Data represent the mean ± s.e.m. (n = 3 performed by triplicate; normalized to control GFP cells; ^***p < 0.001, t-test, compared to the GFP group). (C) Whole-mounted diaphragms of E17.5 mice were immunostained to detect Fzd9 (green) and β-catenin (red). AChR aggregates were stained with αBTX (blue). β-catenin is associated to the sarcolemma of embryonic muscle fibers, including the membrane domains where AChR clusters and Fzd9 are localized. Pictures are representative of at least three experiments performed by triplicate.