miR ‐181a/b downregulation: a mutation‐independent therapeutic approach for inherited retinal diseases
2022; Springer Nature; Volume: 14; Issue: 11 Linguagem: Inglês
10.15252/emmm.202215941
ISSN1757-4684
AutoresSabrina Carrella, Martina Di Guida, Simona Brillante, Davide Piccolo, Ludovica Ciampi, Irene Guadagnino, Jorge García‐Piqueras, Mariateresa Pizzo, Elena Marrocco, Marta Molinari, Georgios Petrogiannakis, Sara Barbato, Yulia Ezhova, Alberto Auricchio, Brunella Franco, Elvira De Leonibus, Enrico Maria Surace, Alessia Indrieri, Sandro Banfi,
Tópico(s)RNA regulation and disease
ResumoArticle4 October 2022Open Access Source DataTransparent process miR-181a/b downregulation: a mutation-independent therapeutic approach for inherited retinal diseases Sabrina Carrella Corresponding Author Sabrina Carrella [email protected] [email protected] orcid.org/0000-0002-3302-1698 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Ecosustainable Marine Biotechnology Department, Stazione Zoologica Anton Dohrn, Naples, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Martina Di Guida Martina Di Guida Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Simona Brillante Simona Brillante orcid.org/0000-0003-0481-1997 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Davide Piccolo Davide Piccolo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Ludovica Ciampi Ludovica Ciampi orcid.org/0000-0002-3309-2402 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Irene Guadagnino Irene Guadagnino Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Jorge Garcia Piqueras Jorge Garcia Piqueras orcid.org/0000-0002-2501-6653 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Mariateresa Pizzo Mariateresa Pizzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Validation, Investigation Search for more papers by this author Elena Marrocco Elena Marrocco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Marta Molinari Marta Molinari Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation, Methodology Search for more papers by this author Georgios Petrogiannakis Georgios Petrogiannakis Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Molecular Life Science, Department of Science and Environmental, Biological and Farmaceutical Technologies, University of Campania "Luigi Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Sara Barbato Sara Barbato Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Yulia Ezhova Yulia Ezhova Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Molecular Life Science, Department of Science and Environmental, Biological and Farmaceutical Technologies, University of Campania "Luigi Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Alberto Auricchio Alberto Auricchio orcid.org/0000-0002-0832-2472 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Advanced Biomedicine, University of Naples "Federico II", Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Brunella Franco Brunella Franco orcid.org/0000-0001-5588-4569 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Scuola Superiore Meridionale, School of Advanced Studies, Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute of Biochemistry and Cellular Biology (IBBC), National Research Council (CNR), Monterotondo, Rome, Italy Contribution: Data curation, Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Enrico Maria Surace Enrico Maria Surace orcid.org/0000-0002-2975-942X Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Alessia Indrieri Alessia Indrieri orcid.org/0000-0002-2325-0913 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy Contribution: Conceptualization, Supervision, Writing - review & editing Search for more papers by this author Sandro Banfi Corresponding Author Sandro Banfi [email protected] orcid.org/0000-0002-6541-8833 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sabrina Carrella Corresponding Author Sabrina Carrella [email protected] [email protected] orcid.org/0000-0002-3302-1698 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Ecosustainable Marine Biotechnology Department, Stazione Zoologica Anton Dohrn, Naples, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Martina Di Guida Martina Di Guida Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Simona Brillante Simona Brillante orcid.org/0000-0003-0481-1997 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Davide Piccolo Davide Piccolo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Ludovica Ciampi Ludovica Ciampi orcid.org/0000-0002-3309-2402 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Irene Guadagnino Irene Guadagnino Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Jorge Garcia Piqueras Jorge Garcia Piqueras orcid.org/0000-0002-2501-6653 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Mariateresa Pizzo Mariateresa Pizzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Validation, Investigation Search for more papers by this author Elena Marrocco Elena Marrocco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Marta Molinari Marta Molinari Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation, Methodology Search for more papers by this author Georgios Petrogiannakis Georgios Petrogiannakis Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Molecular Life Science, Department of Science and Environmental, Biological and Farmaceutical Technologies, University of Campania "Luigi Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Sara Barbato Sara Barbato Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation Search for more papers by this author Yulia Ezhova Yulia Ezhova Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Molecular Life Science, Department of Science and Environmental, Biological and Farmaceutical Technologies, University of Campania "Luigi Vanvitelli", Naples, Italy Contribution: Investigation Search for more papers by this author Alberto Auricchio Alberto Auricchio orcid.org/0000-0002-0832-2472 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Advanced Biomedicine, University of Naples "Federico II", Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Brunella Franco Brunella Franco orcid.org/0000-0001-5588-4569 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Scuola Superiore Meridionale, School of Advanced Studies, Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute of Biochemistry and Cellular Biology (IBBC), National Research Council (CNR), Monterotondo, Rome, Italy Contribution: Data curation, Formal analysis, Supervision, Writing - review & editing Search for more papers by this author Enrico Maria Surace Enrico Maria Surace orcid.org/0000-0002-2975-942X Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Alessia Indrieri Alessia Indrieri orcid.org/0000-0002-2325-0913 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy Contribution: Conceptualization, Supervision, Writing - review & editing Search for more papers by this author Sandro Banfi Corresponding Author Sandro Banfi [email protected] orcid.org/0000-0002-6541-8833 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Sabrina Carrella *,*,1,2, Martina Di Guida1,3, Simona Brillante1, Davide Piccolo1, Ludovica Ciampi1, Irene Guadagnino1,3, Jorge Garcia Piqueras1,3, Mariateresa Pizzo1, Elena Marrocco1, Marta Molinari1, Georgios Petrogiannakis1,4, Sara Barbato1, Yulia Ezhova1,4, Alberto Auricchio1,5, Brunella Franco1,6,7, Elvira De Leonibus1,8, Enrico Maria Surace6, Alessia Indrieri1,9 and Sandro Banfi *,1,3 1Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy 2Ecosustainable Marine Biotechnology Department, Stazione Zoologica Anton Dohrn, Naples, Italy 3Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy 4Molecular Life Science, Department of Science and Environmental, Biological and Farmaceutical Technologies, University of Campania "Luigi Vanvitelli", Naples, Italy 5Medical Genetics, Department of Advanced Biomedicine, University of Naples "Federico II", Naples, Italy 6Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy 7Scuola Superiore Meridionale, School of Advanced Studies, Naples, Italy 8Institute of Biochemistry and Cellular Biology (IBBC), National Research Council (CNR), Monterotondo, Rome, Italy 9Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan, Italy *Corresponding author. Tel: +39 081192830628; E-mail: [email protected]; [email protected] *Corresponding author. Tel: +39 081192830628; E-mail: [email protected] EMBO Mol Med (2022)14:e15941https://doi.org/10.15252/emmm.202215941 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene-specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR-181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR-181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno-associated viral vector (AAV) that harbors a miR-181a/b inhibitor (sponge) sequence (AAV2/8-GFP-Sponge-miR-181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO-P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR-181a/b downregulation modulates the level of the mitochondrial fission-related protein Drp1 and rescues the mitochondrial fragmentation in RHO-P347S photoreceptors. Overall, these data support the potential use of miR-181a/b downregulation as an innovative mutation-independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene-specific therapeutic approaches. Synopsis The application of gene-specific approaches for the treatment of inherited retinal diseases (IRDs) is hampered by their broad genetic heterogeneity. Modulation of the expression of miR-181a/b in the retina represents a promising gene-independent therapeutic strategy for these conditions. miR-181a/b downregulation slows down retinal degeneration in an in vivo model for a dominant form of IRD, i.e., the transgenic RHO-P347S mouse. miR-181a/b downregulation ameliorates the retinal phenotype of an animal model for a recessive form of IRD, i.e., the rd10 mouse. Photoreceptor cells of RHO-P347S mice show an early mitochondrial dysfunction that is counteracted by miR-181a/b downregulation. The paper explained Problem Inherited retinal dystrophies (IRDs) are a group of genetic disorders affecting the retina characterized by photoreceptor cell death, progressive loss of vision and blindness. Their high genetic heterogeneity, with over 250 different causative genes, represents an important limitation to the development of gene therapy approaches that can be applied to a significant number of patients. Therefore, the establishment of therapeutic approaches for IRDs independent on the genetic defect and aimed at slowing down disease progression represents a high priority. Results Here, we show that the inhibition in the retina of two small RNA molecules, namely microRNAs miR-181a and miR-181b (miR-181a/b), exerts a beneficial effect in vivo in different models of IRDs, by delaying disease progression. We also observed a beneficial effect on mitochondria defects observed in these IRDs models. Impact Our findings represent a solid proof-of-principle of the usefulness of miR-181a/b inhibition to counteract disease progression in IRDs and pave the way toward the development of an innovative gene-independent therapeutic strategy for retinal diseases with mitochondrial involvement. Introduction Inherited retinal diseases (IRDs) are among the most prevalent causes of vision loss/blindness of genetic origin in the working-age population. They encompass a variety of different clinical subtypes such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and macular diseases. IRDs display extensive genetic heterogeneity with more than 250 causative genes identified thus far (https://sph.uth.edu/retnet/disease.htm). Gene-replacement therapies are proving to be effective in the treatment of IRDs caused by loss-of-function mutations as exemplified by the recent approval of Voretigene Neparvovec for the treatment of conditions caused by mutations in the RPE65 gene (Ledford, 2017; Apte, 2018). Unfortunately, gene replacement cannot be effectively applied to IRDs caused by gain-of-function mutations, in which the silencing of the mutated allele is primarily required. Moreover, the high genetic heterogeneity of IRDs significantly limit the set up of gene/mutation-specific approaches that can be applied to a large fraction of patients (Carrella et al, 2020). Gene/mutation-independent therapeutic strategies that aim at decreasing and/or delaying cell death in the affected retina, independently of the primary genetic defect, constitute a valid and/or complementary alternative to gene-based approaches (Carrella et al, 2020). Degeneration of photoreceptors (PRs) is the common landmark in IRDs even though a precise knowledge of the underlying molecular and cellular events is still far from reach. Among the most relevant pathways involved, mitochondrial dysfunction, neuroinflammation, and microglia activation are known to worsen PR death and accelerate disease progression (Ambati et al, 2013; Cuenca et al, 2014; Zhao et al, 2015; Lefevere et al, 2017; Mirra & Marfany, 2019; Carrella et al, 2021). Targeting key effectors that impact on common dysregulated pathways in neuronal damage during disease progression hold great promise for therapeutic purposes (Indrieri et al, 2019; Carrella et al, 2020). In the latter respect, microRNAs (miRNAs), given their ability to simultaneously modulate multiple molecular pathways commonly associated with disease pathogenesis and progression, constitute appealing targets toward the design of gene/mutation-independent therapeutic strategies (Askou et al, 2018). We recently identified miR-181a and miR-181b (miR-181a/b) as possible therapeutic targets for mitochondrial-related retinal diseases (Indrieri et al, 2019). MiR-181a/b are highly expressed in the central nervous system (CNS) and in different retinal cell types (Busskamp et al, 2014; Indrieri et al, 2019). They are part of the miR-181 miRNA family and are localized in two distinct genomic clusters in mammals (miR-181a/b-1 and miR-181a/b-2). The mature forms of miR-181a and miR-181b produced by both clusters show identical sequences. Furthermore, the "seed" sequence, that is, the domain hypothesized to play the most relevant role in target recognition, is identical between miR-181a and miR-181b (Indrieri et al, 2020). Several mitochondrial-related transcripts are direct targets of miR-181a/b, for example, Bcl2, Mcl1, and Park2 (Ouyang et al, 2012; Cheng et al, 2016). We recently demonstrated that miR-181a/b control mitochondrial turnover and function in vivo by targeting genes involved in mitochondrial biogenesis, function, and clearance, as well as in reactive oxigen species (ROS) detoxification (i.e., Tfam, Nrf1, Cox11, CoQ10B, and Prdx3) (Indrieri et al, 2019; Barbato et al, 2021). Genetic inactivation of miR-181a/b-1 leads to increased levels of mitochondrial biogenesis and mitophagy in the retina of mouse models of primary mitochondrial dysfunction leading to strong protection from neuronal cell death and to significant amelioration of the disease phenotype (Indrieri et al, 2019). In particular, we showed that genetic inactivation of miR-181a/b-1 protects retinal ganglion cells (RGCs) and ameliorates visual function in different mouse models of Leber's hereditary optic neuropathy (LHON) strongly indicating that these miRNAs may represent effective therapeutic targets for this disease (Indrieri et al, 2019). Notably, we also demonstrated that miR-181a/b-1 ablation did not cause per se neither abnormalities in retinal morphology nor alteration of retinal function, thus supporting the safety of miR-181a/b downregulation in the retina (Indrieri et al, 2019). Over the last decades, evidence linking mitochondrial dysfunction to IRDs is increasing (Carrella et al, 2021). The outer retina is known to have a high metabolic demand, which is associated with abundance of mitochondria in the retinal pigment epithelium (RPE) and PRs. The latter cells are under constant environmental challenges and are highly prone to oxidative stress, a major contributor to retinal degenerations and hence IRDs (Lefevere et al, 2017; Mirra & Marfany, 2019; Jiang et al, 2022). Importantly, since mitochondrial dysfunction seems to be an early event in IRDs, approaches targeting basic mitochondrial functions hold great therapeutic promise (Lefevere et al, 2017; Carrella et al, 2021). Here, we tested the possible neuroprotective effect of miR-181a/b downregulation in IRDs. We demonstrated that miR-181a/b downregulation protects PRs from death and ameliorates their morphology, resulting in improvement of visual function in two different IRD models. We also observed amelioration of mitochondria morphology in PRs, along with a concomitant downregulation of the mitochondrial fission protein Drp1 mediated by the activation of the JAK2/STAT3 pathway. Our data unveil a novel molecular mechanism by which miR-181a/b regulate mitochondrial morphology and function in diseased conditions and identified an AAV-mediated sponge strategy to efficiently downregulate miR-181a/b in vivo. We demonstrated that miR-181a/b downregulation could be effective in ameliorating retinal function across different IRD models with a mutation/gene-independent mechanism, thus providing a novel potential therapeutic strategy that deserves further development toward clinical application in patients. Results miR-181a/b-1 downregulation slows down retinal degeneration in P347S mice The transgenic line carrying the proline-347 to serine (P347S) mutation in the Rhodopsin (RHO) protein is a model for autosomal dominant (AD) RP. These animals show a reduction in electroretinogram (ERG) amplitudes that correlate with the extent of PR loss (Li et al, 1996). The P347S mutation leads to mis-trafficking of the RHO protein with defective vectorial transport of post-Golgi vesicles, which mostly fail in reaching the nascent disks of the PR outer segment (OS) (Li et al, 1996; Greenwald et al, 2013) and is therefore mis-localized to the outer nuclear layer (ONL) in the transgenic mouse retina (Chadderton et al, 2009; Marrocco et al, 2021; Patrizi et al, 2021). A reduced ERG response in RHO-P347S mice (from now onward termed P347S) can be observed at postnatal day (p) 30 and, albeit severely impaired, can be recorded up to p70–90. To study the effect of miR-181a/b inactivation on RP progression, we crossed miR-181a/b-1+/− (Henao-Mejia et al, 2013; Indrieri et al, 2019) with P347S+/+ mice to obtain, in the same litters, P347S+/−/miR-181a/b-1+/+ and P347S+/−/miR-181a/b-1+/− animals, termed P347S and P347S/miR-181a/b-1+/−, respectively (see Materials and Methods). Most of the analyzed miR-181a/b targets (i.e., Cox11, Erk2, Mcl1, Bcl2, Nrf1, Coq10b, Atg5, Xiap, Pgc1a, Park2, and Prdx3) displayed increased transcript levels in P347S/miR-181a/b-1+/− versus P347S eyes, as measured by quantitative (q)RT–PCR (Fig 1A). Figure 1. miR-181a/b-1 downregulation slows down retinal degeneration in P347S mice at p30 A. qRT–PCR analysis reveals increased levels of most of the miR-181a/b targets in the eyes of P347S/miR-181a/b+/− versus P347S animals. N = 4 animals/genotype. B–C′. Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/−. D, E. TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/−. F. Quantification of immunofluorescence analysis in (B–C′) (N = 4 eyes P347S and N = 6 eyes P347S/ miR-181a/b+/−). G. Quantification of TUNEL analysis in (D, E) (N = 3 eyes/genotype). Data information: Scale bars are 50 μm. Data are presented as mean ± SEM. Student's t-test unpaired. [Colour figure can be viewed at wileyonlinelibrary.com] Download figure Download PowerPoint Cell death was analyzed in the retinas of P347S and P347S/miR-181a/b-1+/− animals by active caspase-3 staining and TUNEL assays at p30. A significant decrease of cell death in the ONL of P347S/miR-181a/b-1+/− with respect to P347S was observed (Fig 1B–G). At the same time point, we also detected a significant improvement of ERG responses (both a-waves and b-waves) registered in scotopic conditions in P347S/miR-181a/b-1+/− compared with P347S eyes (Fig 2A–C). Figure 2. miR-181a/b-1 downregulation ameliorates the retinal phenotype in P347S mice at p30 A–C. ERG response [a-wave in (A) and b-wave in (B)], recorded in scotopic conditions, of WT, P347S and P347S/miR-181a/b+/− animals at p30; N ≥ 18 eyes. Data are presented as mean ± SD. Two-way ANOVA test. Representative curves are reported in (C). D–P. Immunofluorescence analysis showed amelioration of Rhodopsin localization (D–F′; white arrowheads); C-arrestin expression (G–I′) and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J–L′); and Phalloidin staining (M–O″) in P347S/miR-181a/b+/− versus P347S eyes at p30. (M″–O″) show higher magnification of (M–O). Scale bars 25 μm in (D–O′); 5 μm in (M″–O″). Fluorescence densitometry quantification of each staining is reported in (P), N = 3 eye/genotype for each staining. WT versus P347S P-values are reported in red, P347S versus P347S/miR-181a/b+/− P-values are reported in black. Data are presented as mean ± SEM. Student's t-test, unpaired. [Colour figure can be viewed at wileyonlinelibrary.com] Download figure Download PowerPoint Although we did not observe significant differences in ONL thickness at p30 (Fig EV1A–D), the expression of the PR markers Rhodopsin (Fig 2D–F′), C-Arrestin (Fig 2G–I′), and Recoverin (Fig EV1E–H) was better preserved in P347S/miR-181a/b-1+/− eyes as compared with P347S controls. In particular, we noticed amelioration of: (i) Rhodopsin distribution, which displayed a lower extent of retainment in the ONL (Fig 2D–F′ and P); (ii) structure of cone and rod OS, as revealed by PNA staining (Fig 2J–L′) and by measurement of OS length on electron microscopy (EM) images (Fig EV1I–K); and (iii) inner segment (IS) structures, as highlighted by Phalloidin staining (Fig 2M–P). Figure 3. The effect of miR-181a/b-1 downregulation in P347S mice is still detectable at p90 A–C. ERG response [a-wave in (A) and b-wave in (B)], recorded in scotopic conditions, of P347S and P347S/miR-181a/b+/− animals at p90; N ≥ 20 eyes/genotype. Data are presented as mean ± SD. Two-way ANOVA test. Representative curves are reported in (C). D–L. Immunofluorescence analysis of Rhodopsin (D–E′), C-arrestin (F–G′), PNA (H–I′); and Phalloidin staining (J–K″) in P347S and P347S/miR-181a/b+/− retinas at p90. (J″–K″) show higher magnification of (J–K). Scale bars 25 μm in (D–K′); 10 μm in (J″–K″). Fluorescence densitometry quantification of each staining is reported in (L), N = 3 eye/genotype/staining. Data are presented as mean ± SEM. Student's t-test, unpaired. M. Measurement of P347S and P347S/miR-181a/b+/− ONL thickness (N ≥ 3 eyes/genotype; Data are presented as mean ± SEM. Student's t-test, unpaired). N. Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles/degree. Visual acuity is preserved in P347S/miR-181a/b+/− animals with respect to P347S (N = 4 animals/genotype). Data are presented as mean ± SEM. Student's t-test, unpaired. [Colour figure can be viewed at wileyonlinelibrary.com] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effects of miR-181a/b-1 downregulation on PRs in P347S mice at p30 A–D. DAPI staining of P347S (A) and P347S/miR-181a/b+/− (B) retinas highlighted no difference in ONL thickness and number of nuclei in the ONL, as quantified in (C) and (D). N = 3 eyes/genotype. Data are presented as mean ± SEM. Student's t-test, unpaired. Scale bars are 50 μm in (A, B). E–H. Immunofluorescence analysis showed amelioration of Recoverin staining in the ONL and the Inner Plexiform Layer (IPL) in P347S/miR-181a/b+/− versus P347S eyes at p30. Scale bars 25 μm. Fluorescence densitometry quantification of Recoverin staining is reported in (H), N = 3 eye/genotype. WT versus P347S P-values are reported in red, P347S versus P347S/miR-181a/b+/− P-values are reported in black. Data are presented as mean ± SEM. Student's t-test. I–K. Electron
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