Effect of PKC on stability of AChR pools at the NMJ in vivo

Previous studies have reported that PKC is involved in the stability of AChRs [11,12,14,22,23]. In this work, we wanted to know which steps of AChR trafficking are regulated by PKC activity at the mature NMJs of living mice. To address this, we first tested whether activation or inhibition of PKC has any effect on the removal of AChRs from postsynaptic sites. To examine this, AChRs on the sternomastoid muscle were labeled with a non-saturating dose of biotinylated α‑bungarotoxin (BTX-biotin) followed by (green) streptavidin-Alexa Fluor4888 (strept-Alexa488) to saturate all biotin sites (see Methods). Four days later, the sternomastoid muscle was exposed and superficial synapses were imaged (time 0) (Figure 1A). The sternomastoid was bathed with PKC inhibitor, calphostin C, continuously for 7 hours and the same synapses were then re-imaged. The loss of fluorescence intensity from NMJs was assayed and compared with untreated synapses. In muscles treated with calphostin C, fluorescence intensity of pre-existing AChRs (not yet internalized) decreased by only 4% (96 ± 6% of original fluorescence; n = 33 NMJs, 3 mice), compared to untreated muscles (p < 0.001), where the fluorescence intensity decreased by 12% (88 ± 5% of original fluorescence; n = 19 NMJs, 3 mice) (Figure 1 B, C). In contrast, in muscles treated with PKC activator, phorbol-12-myristate-13-acetate (PMA), a widely used PKC activator [19,23], pre-existing AChRs fluorescence decreased significantly to 82 ± 9% (n = 39 NMJs, 5 mice), compared to untreated muscles (p < 0.05). (Figure 1 B, C).

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larger image TIFF original image Download: Figure 1. PKC activation accelerates the removal of receptors from synaptic sites in vivo. A, Labeling protocol for assessing the removal of preexisting AChRs from the postsynaptic membrane. Sternomastoid muscles were labeled with biotinylated α-bungarotoxin (BTX-biotin)/Alexa Fluor 488-streptavidin (strept-Alexa488; green). Four days later, superficial synapses were then imaged (time 0) and the sternomastoid muscles were bathed with or without PKC pharmacological agents for 7 h. At the end of the experiment, the same synapses were then imaged. B, Examples of control, and neuromuscular junctions treated with PKC inhibitor calphostin C (CC) and PKC activator phorbol-12-myristate-13-acetate (PMA), that were imaged at time 0 and 7 h later. The total fluorescence intensity of labeled preexisting AChRs was expressed as 100% at the time 0 and 7 hours later. Pseudo-color images provide a linear representation of the density of AChRs. Note that PKC inhibition with CC largely prevents the removal of preexisting AChRs while PKC activation accelerates their loss from postsynaptic membrane. C, Histogram summarizes the amount of preexisting receptors present at synaptic sites, obtained from many junctions by the approach shown in B. Each bar represents the mean percentage of original fluorescence intensity ± SD. D, Labeling method to analyze the insertion of recycled AChRs into the postsynaptic membrane. Sternomastoid muscles were labeled with BTX-biotin/strept-Alexa488; green. Four days later, muscles were bathed again with a saturating dose of strept-Alexa594, red, to selectively label the recycled receptors that had lost their initial strept-Alexa488 tag, while retaining BTX-biotin during the process of internalization and reinsertion. Superficial synapses were then imaged (time 0) and the sternomastoid muscles were bathed with PKC activators and inhibitors. At the end of the experiment, a second saturating dose of strept-Alexa594 was added to label receptors that have been recycled during the treatment. E, Example of control, and neuromuscular junctions treated with PKC inhibitor calphostin C (CC) and PKC activator PMA, that were imaged at time 0 and 8 h later. The total fluorescence intensity of labeled recycled AChRs was expressed as 100% at the time 0 and the fluorescence intensity 8 h later was compared with the fluorescence intensity of the synapse at the previous view. Note that PKC inhibition with CC increases the fluorescence intensity of recycled AChRs while PKC activation with PMA decreases their recycling. F, Histogram summarizes the amount of recycled receptors present at synaptic sites, obtained from many junctions by the approach shown in D. Each bar represents the mean percentage of original fluorescence intensity ± SD.*, p < 0.05; ***, p < 0.001. https://doi.org/10.1371/journal.pone.0081311.g001

Next, we asked whether PKC also affects the normal rate of recycling of previously internalized AChRs into the postsynaptic membrane. To this end, AChRs on the sternomastoid muscle were sequentially labeled with BTX-biotin, followed by a saturating dose of strept-Alexa488, as described in our previous work [3,24]. Four days later, recycled receptors were specifically labeled by adding (red) streptavidin-Alexa594 (strept-Alexa594) to the sternomastoid muscle (strept-Alexa594 binds to receptors that have lost their initial strept-Alexa488 tag while retaining BTX-biotin) [24]. Superficial synapses were imaged immediately (time 0), and the sternomastoid muscle was then bathed with calphostin C, a highly specific PKC blocker, to inhibit PKC [25,26] for the duration of the experiment (7 hours). At the end of the experiment, a second dose of strept-Alexa594 was added to label recycled receptors that had been inserted during the treatment of muscles and the same synapses were imaged for a second time (Figure 1 D). The fluorescence intensity of labeled recycled AChRs was measured before and after treatment. Quantification of recycled AChRs shows that after 7 hours of calphostin C treatment, the fluorescence intensity increased to 114 ± 8% (n = 57 NMJs, 7 mice) of their original fluorescence at time 0 (normalized at 100%) compared to untreated synapses where fluorescence remains unchanged, as previously described by Bruneau et al. [24] (102 ± 3%, n = 15 NMJs, p < 0.001, 3 mice) (Figure 1 E, F). As a second test of PKC inhibition, we used staurosporine (100 nM), a moderately potent PKC blocker, and found that the re-insertion of recycled AChRs at synaptic sites after 7 hours of treatment was also increased, albeit slightly less than with calphostin C (fluorescence intensity of recycled receptors was 106 ± 5% (n = 17 NMJs, 4 mice) versus untreated synapses, 99 ± 3% (n = 21 NMJs, 4 mice, p < 0.001).

The observation that PKC inhibition promotes the recycling of AChRs into synaptic sites prompted us to examine whether activation of PKC would depress the recycling of AChR. AChRs were labeled as described above, and four days later, the sternomastoid muscle was treated with PMA, and 7 hours after treatment, recycled receptors that had been inserted during the treatment of muscles were assessed. Quantification of fluorescently labeled recycled AChRs shows that the density of recycled receptors in muscles treated with PKC activator was significantly decreased (91 ± 7% of original fluorescence; n = 31 NMJs, 5 mice) when compared to untreated synapses (102 ± 3% of original fluorescence; n = 15 NMJs, 3 mice) (Figure 1 E, F).

Given the involvement of PKC activity on AChR recycling, we asked whether the increase of the recycled pool is due to an enhanced stability of recycled receptors in the membrane and/or to the promotion of the insertion of new recycled receptors. To distinguish between these two possibilities, AChRs on the sternomastoid muscles were labeled with BTX-biotin/strept-Alexa488 and four days later, the sternomastoid muscle was exposed and bathed with strept-Alexa594 to specifically label AChRs that had recycled after the initial labeling, and then the superficial synapses were imaged. The muscles were then treated with either PKC inhibitor calphostin C or PKC activator PMA for 7 hours; the same synapses were re-imaged, and their fluorescence intensity was measured. The muscles were bathed again with a second dose of strept-Alexa594, and the same synapses were imaged for a third time (Figure 2 A). Quantification of recycled AChR loss treated with calphostin C showed that the loss of fluorescence was largely prevented, as only 8% of labeled AChR was lost (92 ± 5% of original fluorescence; n = 9 NMJs, 3 mice) (Figure 2 D, E), compared to 19% in non-treated muscles (p < 0.05; 81 ± 3% of original fluorescence; n = 11 NMJs, 3 mice) (Figure 2 B, C). At the same synapses, the number of recycled receptors that had been inserted during the treatment was about 19% (111% - 92%) of the original fluorescence (up to 111 ± 7%; n = 9 NMJs, 3 mice) (Figure 2 D, E), similar (p > 0.05) to the 17% (98% - 81%) increase in control NMJs (up to 98 ± 2%; 11 NMJs, 3 mice) (Figure 2 B, C). In contrast, when muscles were treated with PKC activator PMA, the loss of recycled AChR was significantly increased (p < 0.001) to 34% of the original fluorescence (66 ± 6%; n = 8 NMJs, 3 mice) (Figure 2 F, G), compared to 19% in non-treated muscles. When the number of receptors recycled during the treatment was assessed, it was 17% (83% - 66%) of the original fluorescent pool (up to 83 ± 11%; n = 8 NMJs, 3 mice), not significantly different (p > 0.05) from the 17% (98% - 81%) in non-treated muscles. These results suggest that PKC regulates the AChR recycled pool by reducing the half-life of recycled receptors in the postsynaptic membrane.