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. 2014 Mar 24;9(3):e93140.
doi: 10.1371/journal.pone.0093140. eCollection 2014.

The role of muscle microRNAs in repairing the neuromuscular junction

Gregorio Valdez  1 , Mary P Heyer  2 , Guoping Feng  3 , Joshua R Sanes  4
Affiliations

The role of muscle microRNAs in repairing the neuromuscular junction

Gregorio Valdez et al. PLoS One. .

Abstract

microRNAs have been implicated in mediating key aspects of skeletal muscle development and responses to diseases and injury. Recently, we demonstrated that a synaptically enriched microRNA, miR-206, functions to promote maintenance and repair of the neuromuscular junction (NMJ); in mutant mice lacking miR-206, reinnervation is impaired following nerve injury and loss of NMJs is accelerated in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we asked whether other microRNAs play similar roles. One attractive candidate is miR-133b because it is in the same transcript that encodes miR-206. Like miR-206, miR-133b is concentrated near NMJs and induced after denervation. In miR-133b null mice, however, NMJ development is unaltered, reinnervation proceeds normally following nerve injury, and disease progression is unaffected in the SOD1(G93A) mouse model of ALS. To determine if miR-206 compensates for the loss of miR-133b, we generated mice lacking both microRNAs. The phenotype of these double mutants resembled that of miR-206 single mutants. Finally, we used conditional mutants of Dicer, an enzyme required for the maturation of most microRNAs, to generate mice in which microRNAs were depleted from skeletal muscle fibers postnatally, thus circumventing a requirement for microRNAs in embryonic muscle development. Reinnervation of muscle fibers following injury was impaired in these mice, but the defect was similar in magnitude to that observed in miR-206 mutants. Together, these results suggest that miR-206 is the major microRNA that regulates repair of the NMJ following nerve injury.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of miR-133b at the neuromuscular junction.
(A) Schematic of phrenic nerve innervation and location of NMJs in the mouse diaphragm. Dotted box (A) indicates the region examined using in situ hybridization (B, C). (B, C) Whole-mount miRNA in situ hybridization of P0 diaphragm with antisense (B) but not scrambled (C) DIG-labeled LNA probes shows restriction of miR-133b signal to the central synaptic band. (D) Northern blot analysis of mature miR-133b expression in synaptic (+) and extrasynaptic (−) regions of P21 mouse diaphragm. (E) Intensities of bands in (D), normalized to tRNA, show synaptic enrichment of miR-133b. Error bars indicate SEM. (F) Northern blot analysis of mature miR-133b expression during development of postnatal tibialis anterior muscle.
Figure 2
Figure 2. Normal NMJ development in miR-133b knockout mice.
(A) Immunofluorescence staining of axonal neurofilaments and vesicular synaptophysin (green) and BTX staining of postsynaptic nAChRs (red) to visualize axons innervating synaptic sites. Filled white arrowheads, NMJs with multiple axon innervation; empty arrowheads, retraction bulbs. Scale bar  = 20 μm. (B) The proportion of sternomastoid NMJs with multiple innervation decreases at a similar rate in control and knockout mice. (C) Proportion of developing sternomastoid NMJs with single, double or triple innervation is similar in control and knockout mice.
Figure 3
Figure 3. MiR-133b does not regulate muscle reinnervation or ALS disease progression.
(A) Semi-quantitative RT-PCR of cDNA from control or denervated hindlimb muscle 2 and 4 days after unilateral sciatic nerve cut. Levels of pre-miR-133b and AChRγ increase dramatically in denervated muscle, while levels of pre-miR-133a-1, pre-miR-133a-2 and GAPDH are unchanged, suggesting differential regulation of miR-133a and miR-133b. (B–E) Analysis of muscle reinnervation in tibialis anterior muscle from control (B) and miR-133b null mice (C) 3 weeks following nerve cut. (D) Percentage of tibialis anterior NMJs that were reinnervated. (E) Percentage of NMJs that were denerverated, partially reinnervated, or fully reinnervated. (F,G) Analysis of sternomastoid muscle reinnervation 9 days following accessory nerve crush. At least 6 mice were analyzed and 200 NMJs were examined per animal. Error bars indicate SEM. Scale bar  = 20 μm. (H–J) In the SOD1-G93A mouse model for ALS, loss of miR-133b does not exacerbate symptoms; disease onset (H), survival rate (I), and disease progression (J) are unchanged in the absence of miR-133b. Data were obtained from: 8 female, 8 male SOD1G93A; 10 female, 8 male miR-133b+/−;SOD1G93A; and 6 female, 9 male R-133b−/−;SOD1G93A mice. Error bars indicate SEM.
Figure 4
Figure 4. Generation and analysis of miR-206 and miR-133b double knockout mice.
(A) Trans-allelic targeted meiotic recombination was used to generate mice lacking both miR-206 and miR-133b. MiR-206 and miR-133b heterozygous mice each containing a loxP site in place of the miRNA stemloop were bred together and with mice expressing Cre recombinase in the germline. Zygotes produced from sperm that underwent trans-allelic recombination contained one chromosome lacking miR-206 and miR-133b and one chromosome with a miR-206 and miR-133b duplication. These animals were then bred to obtain miR-206 and miR-133b double knockout mice, i.e. 7H4 knockout mice. P1, forward primer upstream of miR-206. P2, reverse primer downstream of miR-133b. (B) PCR using P1 and P2 primers (in A) gives a detectable product (550 bp) only in 7H4 heterozygous and knockout mice, demonstrating that the 7H4 genomic region containing the miR-206 and miR-133b stem loops is completely missing from the 7H4 null allele. (C) PCR using primers specific for the miR-133b allele yields a 600 bp band only when the WT allele is present, but no band for the 7H4 null allele. (D) Quantitative RT-PCR for the stemloop regions of miR-206 and miR-133b. As expected, miR-206 and miR-133b are absent in 7H4 knockout mice.
Figure 5
Figure 5. Development of NMJs in 7H4 mice.
(A–D) Both miR-206 and miR-133b are dispensable for development of the NMJ. There is no obvious difference in the transformation of the postsynapse (stained using f-BTX, red) from a small plaque into a large pretzel between 7H4 knockout (B and D) and control mice of the same age (A and C). The formation of the presynaptic apparatus is also indistinguishable between 7H4 knockout mice and control mice of the same age, visualized using antibodies against synaptotagmin-2, green, and neurofilament, blue, in young animals (A and B) and YFP expressed in motor axons (C and D). Scale bar  = 10 μm for P9 and 20 μm for adult NMJs.
Figure 6
Figure 6. Lack of both miR-206 and miR-133b delays NMJ regeneration.
(A–D) To determine whether both miRNAs, miR-206 and miR-133b (7H4), act in concert to affect muscle reinnervation, the peroneal nerve was crushed in control (A) and 7H4 knockout mice (B) and reinnervation of the extensor digitorium longus was examined 9 days post injury. In 7H4 muscles, the incidence of partially and completely denervated NMJs is higher than that in muscles from control animals (C, D). At least 6 mice were examined per genotype and 50 NMJs per mouse visualized. FI, fully innervated; PI, partially innervated; FD, fully denervated NMJs. Error bar  =  SEM. P-value (*) <0.02. Scale bar  = 50 μm. (E, F) Quantitative mRNA expression of pre-miR-1-1, pre-miR-1-2, pre-miR-133a-1, and pre-miR-133a-2 in EDL (E) and soleus (F) muscle of adult WT (black circles represent individual values and black line the mean) and 7H4 knockout (red circles represent individual values and red line the mean) mice. Gene expression is normalized to Gapdh and results are scaled to the average value of the WT samples.
Figure 7
Figure 7. Mature miRNAs are required to promote muscle reinnervation.
A tdTomato reporter mouse line was crossed with a mouse expressing Cre recombinase from the Parvalbumin locus. Expression of tdTomato in the EDL, a muscle primarily composed of fast-type muscle fibers, (B) and the soleus, a muscle composed mostly of slow-type muscle fibers (A). tdTomato is also found in axons of proprioceptive sensory neurons (A, arrow), which express Parvalbumin. Scale bar  = 240 μm. (D) NMJs in 1 month-old PV-Cre;Dicer mice are similar to those in control animals (C). (F–H) In PV-Cre-Dicer mice, muscle reinnervation is delayed compared to controls (E, G–H) 7 days post nerve crush. At least 4 mice were examined per genotype and 50 NMJs per mouse visualized. Error bars represent SEM. P-value (*) <0.001. Scale bar  = 20 μm. (I–J) Similarly, deletion of Dicer in mature muscle fibers via electroporation of a plasmid expressing Cre recombinase tagged with GFP also slowed down NMJ regeneration. Error bars represent SEM. Scale bar  = 20 μm.
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References

    1. Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22: 389–442. - PubMed
    1. Wu H, Xiong WC, Mei L (2010) To build a synapse: signaling pathways in neuromuscular junction assembly. Development 137: 1017–1033 10.1242/dev.038711 - DOI - PMC - PubMed
    1. Balice-Gordon RJ (1997) Age-related changes in neuromuscular innervation. Muscle Nerve Suppl 5: S83–7. - PubMed
    1. Bruijn LI, Miller TM, Cleveland DW (2004) Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci 27: 723–749. - PubMed
    1. Punga AR, Ruegg MA (2012) Signaling and aging at the neuromuscular synapse: lessons learnt from neuromuscular diseases. Curr Opin Pharmacol 12: 340–346. - PubMed

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