Abstract
The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that “phosphatidylinositol signaling system” and “extracellular matrix receptor interaction” were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.
Highlights
Neuromuscular junction (NMJ) is a prototypic synapse between the spinal motor neuron and the motor endplate (Hirsch, 2007; Ohno et al, 2017)
Transcriptions of the mouse collagen Q (ColQ) gene start from exons 1 and 1a, which are driven by pColQ1 and pColQ1a promoters, respectively (Figure 1A)
The tibialis anterior (TA) and soleus muscles were teased into single fibers to visualize the NMJs. pCMV-Cre expressed Cre throughout TA muscle, and only 12.5% of Cre-positive areas were overlapped with acetylcholine receptor (AChR)-positive areas
Summary
Neuromuscular junction (NMJ) is a prototypic synapse between the spinal motor neuron and the motor endplate (Hirsch, 2007; Ohno et al, 2017). At the NMJ, acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine (ACh) released from the nerve terminal (Ohno et al, 1998; Gaspersic et al, 1999). PColQ1 is active throughout the slow-twitch muscle. PColQ1 starts transcription from Colq exon 1 to generate A4 and A8 forms. PColQ1a is active at the NMJ, but not at the extrajunctional regions, of the fast-twitch muscle. PColQ1a starts transcription from Colq exon 1a to predominantly generate A12 form. Human pColQ1a carries consensus sequences for transcriptional factors E-protein (Ebox, CANNTG), NFAT (GGAAA), c-Ets transcription factor [c-Ets, (C/A)GGA(A/T)], Elk-1, N-box (CCGGAA), and MEF2 (CTAAAAATAA), which play essential roles in muscle-specific and NMJ-specific transcriptional activities (Lee et al, 2004)
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