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  • AON successfully alleviated the myotonic phenotype in

    2020-02-19

    AON successfully alleviated the myotonic phenotype in DM model mice [18], [23]. To screen for an optimal AON sequence, we used 25-mer phosphorothioate 2′ O-methyl RNA molecules that covered the exon 6B or exon 7A. Unexpectedly, AON 7A (+63−8) which covers the boundary of intron 7A and exon 7A enhanced the expression of 5–6–7 splicing product. Some AONs designed on exon 7A excluded not only exon 7A but also exon 6B (Fig. 5). Therefore we thought that the exclusion of exons 6B and 7A might be related. AON of exon 7A (+1+25) which was the most successful AON in mice did not enhance 5–6–7 splicing in human. We therefore hypothesise that the differences seen between mice and humans are the result of sequence differences between the human and mouse orthologues of CLCN1, as sequence similarity in exons 5–7 is only 75% according to BLAST analysis. In this study, we described differences in splicing patterns of Clcn1 and CLCN1. These observations are important, as they suggest that results obtained using Clcn1 do not necessarily apply to CLCN1. Subsequent experiments should therefore focus on CLCN1, as opposed to the mouse Clcn1 in investigations on CLCN1 for DM medical treatments.
    Acknowledgements We thank Dr. Yoshihiro Kino for providing the CLCN1 minigene. We also thank Dr. Satoshi Suo, Ms. Kurara Takagane, Ms. Shoin Tei, Mr. Takashi Nagashima, Mr. Ryo Yonezawa and Mr. Hiroshige Ishii for valuable discussions. This work was supported in part by an Intramural Research Grant (MHLW;26-8) for Neurological and Psychiatric Disorders of NCNP, a research grant for Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour and Welfare (MHLW; H26-Sinkeikinn-ippan-004) and a Grant-in-Aid from the MHLW of Japan (to S.I.).
    Introduction Cys-loop ligand-gated chloride LDC000067 sale (LGCCs) are membrane protein complexes composed of five subunits. LGCCs cause chloride ion influx in response to neurotransmitters and thereby stabilize the resting potential to suppress firing action potentials in neurons or muscle cells (Miller and Smart, 2010). Vertebrates have two major LGCCs, γ-aminobutyric acid (GABA)- and glycine-gated chloride channels, whereas insects, such as the fruit fly Drosophila melanogaster, have LGCCs for GABA, l-glutamate (Glu) and histamine (HA) (Cully et al., 1996, ffrench-Constant et al., 1991, Geng et al., 2002, Gisselmann et al., 2002, Witte et al., 2002, Zheng et al., 2002) and the free-living nematode Caenorhabditis elegans has LGCCs for tyramine, dopamine, serotonin, choline and acetylcholine, in addition to those for GABA and Glu (Bamber et al., 1999, Cully et al., 1994, Pirri et al., 2009, Putrenko et al., 2005, Ranganathan et al., 2000, Ringstad et al., 2009). It is interesting that invertebrates have various unique LGCCs. Of these, insect GABA-gated chloride channels (GABACls) are established targets with unique binding sites for insecticides and ectoparasiticides (Ozoe, 2013, Ozoe et al., 2015). Nematode Glu-gated chloride channels (GluCls) represent important targets for the anthelmintic ivermectin. However, LGCCs other than GABACls and GluCls are not pharmacologically well characterized. HA is a biogenic amine present at high concentrations in the retina of insects (Elias and Evans, 1983), whereas it is abundantly found in the stomach, lymph nodes and thymus as a local mediator in vertebrates (Zimmermann et al., 2011). Intense HA-like immunoreactivity is detected in photoreceptor terminals in the first optic neuropil lamina of the compound eye of flies (Nässel et al., 1988). Photoreceptor neurons, which are depolarized by light, are HAergic and presynaptic to large monopolar cells (LMCs) in the lamina of insects, thereby indicating the neurotransmitter role of HA (Hardie, 1987, Stuart et al., 2007). HA is not only the neurotransmitter of photoreceptors but has also been reported to play important roles in mechanosensory reception, temperature preference and sleep in insects (Hong et al., 2006, Melzig et al., 1998).