Niccolò Bacchi1, Andrea Messina1, Verena Burtscher2, Erik Dassi1, Giovanni Provenzano1, Yuri Bozzi3, Gian Carlo Demontis4, Alexandra Koschak5, Michela A Denti6, Simona Casarosa3. 1. Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy. 2. Medical University of Vienna, Center for Physiology and Pharmacology, Department of Neurophysiology and Pharmacology, Vienna, Austria. 3. Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy 3Neuroscience Institute-National Research Council (CNR), Pisa, Italy. 4. Department of Pharmacy, University of Pisa, Pisa, Italy. 5. Medical University of Vienna, Center for Physiology and Pharmacology, Department of Neurophysiology and Pharmacology, Vienna, Austria 5University of Innsbruck, Institute of Pharmacy, Pharmacology and Toxicology, Center for Chemistry and Biomedicine, Innsb. 6. Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy 6Neuroscience Institute-National Research Council (CNR), Padova, Italy.
Abstract
PURPOSE: Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c.2406C→A mutation) and mice (c.2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons. METHODS: Splicing of the targeted α2δ4 (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 L-type calcium channels (CACNA1F). RESULTS: Splicing analysis revealed the presence of a previously unknown splicing isoform of α2δ4 in the retina that truncates the gene open reading frame (ORF) in a similar way as the c.2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c.2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length α2δ4 was able to increase Cav1.4/β3-mediated currents while all other α2δ4 variants did not mediate such effect. CONCLUSIONS: The designed exon-skipping strategy is not applicable because the resulting skipped α2δ4 are nonfunctional. α2δ4 E25b splicing variant is normally present in mouse retina and mimics the effect of c.2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals.
PURPOSE: Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c.2406C→A mutation) and mice (c.2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons. METHODS: Splicing of the targeted α2δ4 (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 L-type calcium channels (CACNA1F). RESULTS: Splicing analysis revealed the presence of a previously unknown splicing isoform of α2δ4 in the retina that truncates the gene open reading frame (ORF) in a similar way as the c.2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c.2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length α2δ4 was able to increase Cav1.4/β3-mediated currents while all other α2δ4 variants did not mediate such effect. CONCLUSIONS: The designed exon-skipping strategy is not applicable because the resulting skipped α2δ4 are nonfunctional. α2δ4 E25b splicing variant is normally present in mouse retina and mimics the effect of c.2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals.
Authors: Vasily Kerov; Joseph G Laird; Mei-Ling Joiner; Sharmon Knecht; Daniel Soh; Jussara Hagen; Sarah H Gardner; Wade Gutierrez; Takeshi Yoshimatsu; Sajag Bhattarai; Teresa Puthussery; Nikolai O Artemyev; Arlene V Drack; Rachel O Wong; Sheila A Baker; Amy Lee Journal: J Neurosci Date: 2018-06-06 Impact factor: 6.167
Authors: Stefanie Geisler; Clemens L Schöpf; Ruslan Stanika; Marcus Kalb; Marta Campiglio; Daniele Repetto; Larissa Traxler; Markus Missler; Gerald J Obermair Journal: J Neurosci Date: 2019-01-25 Impact factor: 6.167