miR‐181a/b downregulation exerts a protective action on mitochondrial disease models
2019; Springer Nature; Volume: 11; Issue: 5 Linguagem: Inglês
10.15252/emmm.201708734
ISSN1757-4684
AutoresAlessia Indrieri, Sabrina Carrella, Alessia Romano, Alessandra Spaziano, Elena Marrocco, Erika Fernández‐Vizarra, Sara Barbato, Mariateresa Pizzo, Yulia Ezhova, Francesca M Golia, Ludovica Ciampi, Roberta Tammaro, Jorge Henao‐Mejia, Adam Williams, Richard A. Flavell, Elvira De Leonibus, Massimo Zeviani, Enrico Maria Surace, Sandro Banfi, Brunella Franco,
Tópico(s)RNA regulation and disease
ResumoResearch Article12 April 2019Open Access Source DataTransparent process miR-181a/b downregulation exerts a protective action on mitochondrial disease models Alessia Indrieri Alessia Indrieri orcid.org/0000-0002-2325-0913 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Search for more papers by this author Sabrina Carrella Sabrina Carrella Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Caserta CE, Italy Search for more papers by this author Alessia Romano Alessia Romano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Alessandra Spaziano Alessandra Spaziano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Elena Marrocco Elena Marrocco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Erika Fernandez-Vizarra Erika Fernandez-Vizarra orcid.org/0000-0002-2469-142X MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Sara Barbato Sara Barbato Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Mariateresa Pizzo Mariateresa Pizzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Yulia Ezhova Yulia Ezhova Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Francesca M Golia Francesca M Golia Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Ludovica Ciampi Ludovica Ciampi Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Roberta Tammaro Roberta Tammaro Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Jorge Henao-Mejia Jorge Henao-Mejia Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Adam Williams Adam Williams The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Department of Genetics and Genomic Sciences, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Richard A Flavell Richard A Flavell Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute of Cellular Biology and Neurobiology "ABT", CNR, Roma, Italy Search for more papers by this author Massimo Zeviani Massimo Zeviani orcid.org/0000-0002-9067-5508 MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Enrico M Surace Enrico M Surace Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy 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", Caserta CE, Italy Search for more papers by this author Brunella Franco Corresponding Author Brunella Franco [email protected] 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 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 Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy Search for more papers by this author Sabrina Carrella Sabrina Carrella Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Caserta CE, Italy Search for more papers by this author Alessia Romano Alessia Romano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Alessandra Spaziano Alessandra Spaziano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Elena Marrocco Elena Marrocco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Erika Fernandez-Vizarra Erika Fernandez-Vizarra orcid.org/0000-0002-2469-142X MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Sara Barbato Sara Barbato Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Mariateresa Pizzo Mariateresa Pizzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Yulia Ezhova Yulia Ezhova Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Francesca M Golia Francesca M Golia Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Ludovica Ciampi Ludovica Ciampi Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Roberta Tammaro Roberta Tammaro Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Jorge Henao-Mejia Jorge Henao-Mejia Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Adam Williams Adam Williams The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA Department of Genetics and Genomic Sciences, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Richard A Flavell Richard A Flavell Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Institute of Cellular Biology and Neurobiology "ABT", CNR, Roma, Italy Search for more papers by this author Massimo Zeviani Massimo Zeviani orcid.org/0000-0002-9067-5508 MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Enrico M Surace Enrico M Surace Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy 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", Caserta CE, Italy Search for more papers by this author Brunella Franco Corresponding Author Brunella Franco [email protected] 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 Search for more papers by this author Author Information Alessia Indrieri1,2,‡, Sabrina Carrella1,3,‡, Alessia Romano1, Alessandra Spaziano1, Elena Marrocco1, Erika Fernandez-Vizarra4, Sara Barbato1, Mariateresa Pizzo1, Yulia Ezhova1, Francesca M Golia1, Ludovica Ciampi1, Roberta Tammaro1, Jorge Henao-Mejia5,6, Adam Williams7,8, Richard A Flavell9,10, Elvira De Leonibus1,11, Massimo Zeviani4, Enrico M Surace1,2,12, Sandro Banfi *,1,3 and Brunella Franco *,1,2 1Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy 2Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy 3Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Caserta CE, Italy 4MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK 5Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA 6Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 7The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 8Department of Genetics and Genomic Sciences, University of Connecticut Health Center, Farmington, CT, USA 9Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA 10Howard Hughes Medical Institute, Chevy Chase, MD, USA 11Institute of Cellular Biology and Neurobiology "ABT", CNR, Roma, Italy 12Present address: Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +39 08119230606; E-mail: [email protected] *Corresponding author. Tel: +39 08119230605; E-mail: [email protected] EMBO Mol Med (2019)11:e8734https://doi.org/10.15252/emmm.201708734 PDFDownload PDF of article text and main figures. 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 Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration. Synopsis MicroRNAs 181a/b is important for mitochondria homeostasis in the retina. miR-181a/b inactivation in different animal models of mitochondrial diseases protects neuronal degeneration and ameliorates the disease phenotype in tested models. miR-181a/b control mitochondrial biogenesis in the retina and their downregulation enhances mitochondrial turnover through the coordinated activation of mitochondrial biogenesis and mitophagy. miR-181a/b inhibition protects neurons from cell death and ameliorates the phenotype of different in vivo models of mitochondrial disease, i.e. such as Microphthalmia with Linear Skin Lesions (MLS) and Leber Hereditary Optic Neuropathy (LHON). miR-181a/b may represent effective gene-independent therapeutic targets for genetically heterogeneous mitochondrial diseases characterized by neuronal degeneration. Introduction Mitochondrial diseases (MDs) represent a relevant group of inherited disorders with a cumulative prevalence of about 1:5,000 individuals (Gorman et al, 2015). They are caused by mutations in either nuclear or mitochondrial genes resulting in oxidative phosphorylation (OXPHOS) impairment, leading to huge variability of symptoms, organ involvement, and clinical course. The clinical manifestations range from dysfunction of single tissue/structures such as the optic nerve in Leber's hereditary optic neuropathy (LHON, MIM535000), to syndromic multi-organ conditions with a prominent involvement of the central nervous system (CNS), such as microphthalmia with linear skin lesions (MLS, MIM309801, 300887, 300952) and Leigh syndrome (LS, MIM256000). Neurons are particularly sensitive to mitochondrial dysfunction due to their highest energy demands, and defects in mitochondrial metabolism may lead to severe energy deficiency, increased reactive oxygen species (ROS), and neuronal death. MDs are biochemically and genetically heterogeneous, and their complexity has so far prevented the development of effective treatments (Sanchez et al, 2016). In the past few years, specific modulation of either mitochondrial biogenesis/dynamics or mitochondrial clearance/quality control has been tested as possible therapeutic strategies in different MD models (Viscomi et al, 2011; Johnson et al, 2013; Cerutti et al, 2014; Civiletto et al, 2015, 2018). In spite of initial promising data, the latter approaches failed to be effective across different MD models [reviewed in Lightowlers et al (2015); Viscomi et al (2015)]. We hypothesize that a synergic and fine modulation of mitochondrial biogenesis and clearance pathways is necessary to ensure a more efficient neuroprotective effect. MicroRNAs (miRNAs) are fundamental fine regulators of gene expression and represent promising therapeutic tools due to their capability of simultaneously modulating multiple molecular pathways involved in disease pathogenesis and progression. Modulation of miRNAs has been applied, with therapeutic purposes, to different disorders and has reached preclinical and clinical stages in specific instances (Broderick & Zamore, 2011; Janssen et al, 2013; Ling et al, 2013; Christopher et al, 2016). miRNAs play a key role in neuron survival, and accumulating evidence indicates that alterations of miRNA-regulated gene networks increase the risk of neurodegenerative disorders (Hebert & De Strooper, 2009). miR-181a and miR-181b (miR-181a/b) belong to a family of miRNAs highly expressed in different regions of brain and retina (Boudreau et al, 2014; Karali et al, 2016) and were recently reported to target genes involved in mitochondrial-dependent cell death (Ouyang et al, 2012; Hutchison et al, 2013; Rodriguez-Ortiz et al, 2014) and autophagy (He et al, 2013; Tekirdag et al, 2013; Cheng et al, 2016). Here, we show that miR-181a/b are involved in global regulation of mitochondrial function by controlling a group of genes involved in mitochondrial biogenesis and function, and redox balance. We demonstrate that downregulation of these two miRNAs protects retinal neurons from mitochondrial dysfunction, and ameliorates the phenotype of three different MD animal models with ocular involvement, indicating that miR-181a/b could be new therapeutic targets for MDs. Results miR-181a/b control mitochondrial turnover Bioinformatic search (Gennarino et al, 2012) allowed us to identify PPARGC1A and NRF1, master regulators of mitochondrial biogenesis (Wu et al, 1999; Finck & Kelly, 2006), COX11 and COQ10B, involved in mitochondrial respiratory chain (MRC) assembly (Carr et al, 2002; Desbats et al, 2015), and PRDX3, an important mitochondrial ROS scavenger (Wonsey et al, 2002), as putative miR-181a/b target genes. Interestingly, quantitative real-time PCR (qPCR) analysis indicated increased levels of all of the above-mentioned transcripts in SH-SY5Y human neuroblastoma cells following miR-181a/b silencing (Fig 1A). To validate the newly predicted miR-181a/b targets, 3′-UTRs of each human gene (PPARGC1A, NRF1, COX11, COQ10B, and PRDX3), including the predicted miR-181 target site, were cloned in the pGL3-TK-luciferase plasmid, downstream the coding region of the luciferase reporter gene. We then tested the ability of transfected mimic-miR-181 to affect luciferase activity. The presence of the 3′-UTR sequence of the analyzed genes inhibited luciferase activity in response to mimic-miR-181 (Fig 1B). In addition, point mutations in the miR-181a/b binding site in the 3′-UTR of each gene abolished luciferase repression, demonstrating that these miRNAs directly and specifically target NRF1, COX11, COQ10B, and PRDX3 (Fig 1B). Direct targeting of PPARGC1A was not validated (Fig 1B), indicating that the upregulation observed by qPCR after miR-181a/b silencing (Fig 1A) could be the result of an indirect effect. Figure 1. miR-181a/b silencing increases mitochondrial biogenesis and mitophagy and protects retinal neurons from FCCP-induced cell death A. qPCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N = 3 independent experiments. B. miR-181-mimic transfection specifically inhibits luciferase activity of constructs containing WT 3′-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N = 6 independent experiments. C. qPCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1−/− versus miR-181a/b-1+/+ animals. n ≥ 5 animals/genotype. D. miR-181a/b-1−/− mice show increased mtDNA content versus miR-181a/b-1+/+ mice as measured by qPCR. N = 4 animals/genotype. E. WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1−/− (-/-) versus miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N ≥ 3 animals/genotype. Please note that all compared bands from +/+ and −/− samples are from the same blots, which were cropped and shown split for the sake of data presentation clarity. F. WB analysis on mitochondrial and cytosolic fractions (left panel) shows increased levels of p62 and Parkin in mitochondrial fraction from the eye of −/− versus +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N = 3 animals/genotype. G. Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N ≥ 7 independent experiments. Data information: P-values were calculated by one-tailed Student's t-test in (A–C, E and F), by two-tailed Student's t-test in (D), and by two-way ANOVA with post hoc Tukey's analysis in (G); error bars are SEM. Source data are available online for this figure. Source Data for Figure 1 [emmm201708734-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Based on the above results, we reasoned that the downregulation of miR-181a/b could stimulate mitochondrial biogenesis and decided to test this hypothesis in vivo. In mammals, miR-181a and miR-181b are organized in two clusters, namely miR-181a/b-1 and miR-181a/b-2, which are localized to different genomic loci. The mature forms of miR-181a-1 and miR-181a-2, as well as those of miR-181b-1 and miR-181b-2, display identical sequences. Furthermore, both miR-181a and miR-181b contain the same "seed" sequence (Ji et al, 2009), i.e., the region that is believed to play the most important role in target recognition (Bartel, 2009). We chose to analyze a mouse model harboring a targeted deletion of the miR-181a/b-1 cluster (Henao-Mejia et al, 2013). This cluster accounts for most of the expression of mature miR-181a/b in the retina, as demonstrated by RNA in situ hybridization, TaqMan assays, and the increase in several previously validated miR-181a/b targets, such as Bcl2, Mcl1, Atg5, Erk2, and Park2 (Ouyang et al, 2012; He et al, 2013; Hutchison et al, 2013; Tekirdag et al, 2013; Rodriguez-Ortiz et al, 2014; Cheng et al, 2016) (Appendix Fig S1, Fig EV1A). Click here to expand this figure. Figure EV1. miR-181a/b-1 deletion leads to increased autophagic flux A. qPCR analysis reveals increased levels of miR-181a/b targets Bcl2, Mcl1, Atg5, Erk2, and Park2 in the eyes of miR-181a/b-1−/− versus miR-181a/b-1+/+ animals. N ≥ 5 animals/genotype. B. WB analysis (left panel) of Lc3-I/Lc3-II and p62 on protein extracts from eyes of animal in fed and starved conditions reveals decreased levels (quantified in the right panel) of both proteins in miR-181a/b-1−/− (−/−) versus miR-181a/b-1+/+ (+/+) mice. N = 2 mice for each genotype and condition. C. qPCR analysis reveals no changes in the Sqstm1 (p62) and Map1lc3b (LC3) transcript levels between miR-181a/b-1−/− and miR-181a/b-1+/+ mouse eyes in both fed and starved conditions. These data indicate that the decreased levels of the autophagy markers Lc3-II and p62 observed by WB analysis are due to increased autophagic flux in miR-181a/b-1−/− eyes. N = 3 for each genotype and condition. Data information: P-values were calculated by one-tailed Student's t-test; error bars are SEM. Source data are available online for this figure. Download figure Download PowerPoint Interestingly, by qPCR, we observed increased expression levels of Nrf1, Cox11, Coq10b Prdx3, and Ppargc1a in the eye of miR-181a/b-1−/− mice (Fig 1C). Upregulation of Nrf1 and Ppargc1a indicates enhanced mitochondrial biogenesis (Wu et al, 1999; Finck & Kelly, 2006). In line with this observation, we detected an increase of mitochondrial DNA (mtDNA), as measured by qPCR (Fig 1D), and of the protein levels of MRC complex subunits (Ndufb11 and CoxIV), mitochondrial matrix (citrate synthase [Cs]), and mitochondrial membranes (Mfn2 and Tim23), as assessed by Western blot (WB) analysis in the eye of miR-181a/b-1−/− mice (Fig 1E). Overall, these data demonstrate that miR-181a/b inactivation stimulates mitochondrial biogenesis in the CNS. It was previously reported that miR-181a/b regulate in vitro the expression of Atg5 and Park2, which are key players in autophagy and mitophagy (Tekirdag et al, 2013; Cheng et al, 2016). Therefore, we investigated whether miR-181a/b inactivation enhances mitophagy in the mouse eye. First, we observed, by qPCR assays, that the transcript levels of the Atg5 and Park2 genes were upregulated in miR-181a/b-1−/− eyes (Fig EV1A). Moreover, we demonstrated, by WB, enhanced recruitment of Park2/Parkin and of the autophagic adaptor Sqstm1/p62 in mitochondrial fractions (Fig 1F), indicating an increase of mitophagy in miR-181a/b-1−/− eyes. We also analyzed general autophagy by evaluating the levels of Sqstm1/p62 and of the autophagic marker Map1lc3b/Lc3-II in total protein extracts. By WB, we observed decreased levels of these two proteins, which is more evident in starved conditions (Fig EV1B), without changes in the corresponding transcript levels (Fig EV1C), indicating an increase in the autophagic flux rate in miR-181a/b-1−/− eyes. Taken together, these results uncover an important role of miR-181a/b in the regulation of mitochondrial turnover through the coordination of mitochondrial biogenesis and clearance in vivo. miR-181a/b inhibition protects neurons from cell death and ameliorates the phenotype of in vivo models of MLS syndrome Increased mitochondrial biogenesis and clearance were previously shown to exert protective effects in mitochondrial dysfunction (Viscomi et al, 2011; Johnson et al, 2013; Cerutti et al, 2014; Civiletto et al, 2015, 2018). We therefore decided to verify whether miR-181a/b inactivation could protect cells from mitochondrial damage. First, we verified whether miR-181a/b downregulation protects SH-SY5Y neuron-like cells from FCCP, a potent OXPHOS uncoupler. In control cells, 6 hours (h) of FCCP treatment induced a significant increase in the extent of cell death. Interestingly, miR-181a/b-silenced cells did not display any differences in cell death after FCCP treatment (Fig 1G), indicating that miR-181a/b silencing protects cells from mitochondrial damage. Based on the above results, we decided to evaluate the neuroprotective effect of miR-181a/b inactivation in in vivo models of MDs. Toward this goal, we examined the consequences of miR-181a/b downregulation in two fish models for a rare inherited form of MD, the MLS syndrome. MLS is a neurodevelopmental disorder characterized by microphthalmia, brain abnormalities, and skin defects in heterozygous females and in utero lethality in hemizygous males (Indrieri & Franco, 2016). The disease is due to mutations in key players of the MRC, such as the holocytochrome c-type synthase (HCCS), involved in complex III function (Bernard et al, 2003; Wimplinger et al, 2006; Indrieri et al, 2013), and COX7B, the 7B subunit of cytochrome c oxidase (MRC complex IV) (Indrieri et al, 2012). We previously generated two medakafish (Oryzias latipes) models of MLS by knocking down, using a Morpholino(MO)-based approach, hccs or cox7B expression (Indrieri et al, 2012, 2013). Both models (hccs-MO and cox7B-MO) showed a severe microphthalmic and microcephalic phenotype due to increased cell death in the CNS (Indrieri et al, 2012, 2013, 2016). In medaka, the mature forms of miR-181a and miR-181b are perfectly conserved with respect to their mammalian counterparts, in terms of both sequence identity (100%) and pattern of expression in the retina and brain (Carrella et al, 2015). We found that MO-mediated silencing of miR-181a/b in medaka leads to increased levels of the majority of targets involved in mitochondrial biogenesis and function, and in autophagy (Fig EV2). Interestingly, downregulation of miR-181a/b in either of the above-mentioned MLS medaka models led to a notable reduction of cell death in the eye and brain, as shown by TUNEL and caspase activation assays (Fig 2A–C). Accordingly, miR-181a/b downregulation resulted in full rescue of the disease phenotype in about 50% of both hccs (Fig 3A–C and M) and cox7B morphants (Fig 3G–I and N). Notably, MO-mediated silencing of miR-181a/b did not cause any obvious morphological alteration in the controls (Fig EV3A and B). These data show that the downregulation of miR-181a/b ameliorates the phenotype in both MRC complex III and IV defective models, indicating that the protective effect of miR-181a/b silencing is gene-independent. Click here to expand this figure. Figure EV2. miR-181a/b knockdown in medaka leads to upregulation of genes involved in mitochondrial function and autophagyqPCR carried out on total RNA extracted from whole miR-181a/b-MOs medaka embryos to analyze the transcript levels of the miR-181a/b targets involved in mitochondrial-dependent cell death (bcl2, mcl1), autophagy (atg5, erk2), and mitochondrial biogenesis and function (cox11, coq10b, prdx3) and of the indirect target ppargc1a. N = 3. P-values were calculated by one-tailed Student's t-test; error bars are SEM. Download figure Download PowerPoint Figure 2. miR-181a/b inhibition counteracts cell death in MLS medakafish models A. TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7B-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7B-MO-injected embryos. Scale bars are 20 μm. B. TUNEL-positive cells/eye. n ≥ 5 eyes for each model. C. Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7B-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7B-MO-injected embryos. n ≥ 5 embryos for each model. Data information: P-values were calculated by analysis of deviance for negative binomial generalized linear model in (B) and by one-way ANOVA with post hoc Tukey's analysis in (C); error bars are SEM. Download figure Download PowerPoint Figure 3. miR-181a/b downregulation ameliorates the phenotype of MLS medakafish models A–L. Representative images of st30 medaka embryos injected with hccs-MO (B) and cox7B-MO (H) alone or co-injected with miR-181a/b-MOs (C, I). Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO and cox7B-MO embryos. (D–F, J–L) hccs-MO/miR-181a/b-MO- and cox7B-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059, or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in both hccs-MO/miR-181a/b-MO and cox7B-MO/miR-181a/b-MO embryos (D, J). PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation (E, K). HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model but not in the cox7B-MO/miR-181a/b-MO embryos (F, L). Scale bars are 100 μm. M, N. Percentage of embryos with or without MLS phenotype in the different conditions illustrated in (A–L) in hccs-MO (M) and cox7B-MO (N) embryos. N ≥ 300 embryos/conditions. P-values were calculated by analysis of deviance for generalized linear model; error bars are SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Absence of abnormal phenotypes following MO-mediated silencing of miR-181a/b and drug administration A, B. Embryos injected with miR-181a/b-MO
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