miR-181a/b downregulation exerts a protective action on mitochondrial disease models.

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\u27s 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

miR-181a/b downregulation exerts a protective action on mitochondrial disease models.

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.Italian Fondazione Telethon (grant no. TGM16YGM02 to S. Ban, the Fondazione Roma (grant no. RP‐201300000009 to S. Ban)) and the AFM‐Telethon (grant no. 20685 to B.F.). A.I. received an Umberto Veronesi Fellowship. This research was carried out in the frame of Programme STAR, financially supported by UniNA and Compagnia di San Paolo (Bando STAR, 16‐CSP‐UNINA‐048, to A.I)

Transcriptomic determinants of vertebrate photoreceptors and other sensory cells

The nervous system comprises an immense number of cells that serve highly specialized roles. Accordingly, they possess unique morphological and functional attributes, greatly shaped by specific Alternative Splicing (AS) and Gene Expression (GE) mechanisms. Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light by way of the ciliary outer segment. We discovered a new layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in-vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion, severe photoreceptor defects and blindness. Since this microexon program involves genes of the photoreceptor cilium, we explored whether other ciliated sensory cells display specialized transcriptomes. Multi-layer RNA-seq analysis revealed that cilia in olfactory neurons diverge from other ciliated sensory cells by activating a network of motile cilia genes highly expressed in spermatocytes and regulated by Rfx3. Our results shed light on the transcriptomic specialization and functionality of cilia in sensory cells, uncovering new cell-type specific roles for many genes with implication for a wide spectrum of human diseases.El sistema nervioso comprende una inmensa cantidad de células que cumplen funciones altamente especializadas. En consecuencia, poseen atributos morfológicos y funcionales únicos, moldeados en gran medida por mecanismos específicos de splicing alternativo (AS) y expresión génica (GE). Los fotorreceptores retinales tienen un perfil transcriptómico distinto en comparación con otros subtipos neuronales, lo que probablemente refleja su morfología celular y función únicas en la detección de luz a través del segmento externo ciliar. Descubrimos una nueva capa de esta especialización molecular al revelar que la retina de los vertebrados expresa la mayor cantidad de microexones enriquecidos en tejidos (entre todos los tejidos analizados). Este programa de microexones está regulado por Srrm3, un parálogo del regulador de microexones neurales Srrm4. A pesar de que ambas proteínas regulan positivamente los microexones de la retina in vitro, solo Srrm3 se expresa en gran medida en los fotorreceptores maduros. Su eliminación en el pez cebra resulta en una regulación negativa generalizada de la inclusión de microexones, defectos graves en los fotorreceptores y ceguera. Dado que este programa de microexón involucra genes del cilio de los fotorreceptores, exploramos si otras células sensoriales ciliadas muestran transcriptomas especializados. El análisis multi-layer RNA-seq reveló que los cilios en las neuronas olfatorias se separan de los de otras células sensoriales ciliadas al activar una red de genes de cilios móviles altamente expresados ​​en los espermatozoides y regulados por Rfx3. Nuestros resultados elucidan la especialización transcriptómica y la funcionalidad de los cilios en las células sensoriales, revelando nuevas funciones célula-específicas para muchos genes con implicación en un amplio espectro de enfermedades humanas.Programa de Doctorat en Biomedicin

Table 4.2

  Table 4.2: Primer sequences used in Chapter 4. It contains primer name, sequence and application in the study.</p

Table 5.5

  Table 5.5: Source data for GSEA and Olfactory-Sperm GO analysis All GSEA enrichments were performed through the R package fgsea (v1.10.1), using as input all the features (genes) and their weights assigned by MOFA. The first sheet contains GSEA results for the 250 genes harboring microexons with the most extreme values in the MIC_AS layer, the second contains the GSEA results for all Factor 1 genes for the GE layer. Tha last sheet contains the GO enrichment analysis, performed with the software ClueGO, for the Sperm-Olfactory dataset. GO ID, GO description, corrected P-value, number of genes and associated genes </p

Table 5.1

  Table 5.1: RNA-seq data used in Chapter 5.  SRA and ENA sample identifiers, sample name, read number, library preparation and age are provided for each public RNA-seq file used in this study. The last column includes comments regarding sample isolation and RNA preparation.</p

Table 4.1.xlsx

  Table 4.1: Regulation of retina-enriched exons in SRRM3 OE_ELAVL1 KD and SRRM4 OE_ELAVL1 KD cells. Inclusion of RetMICs among all the cell lines and experiments included in Chapter 4. Columns 1-4: name, event VastID, event genomic coordinate, exon length. Columns 5-10: RetMICs PSI in cells expressing an Empty Vector (EV), EV+scramble sequence, SRRM3+ELAVL1 KD, SRRM3+scramble sequence, SRRM4+ELAVL1 KD and SRRM4+scramble. ΔPSI between the different OE and KD conditions is provided in columns 10-13. Only events with sufficient read coverage are shown, to reliably estimate inclusion levels. </p

Table 4.2

  Inclusion of RetMICs among all the cell lines and experiments generated in Chapter 4. Columns 1-4: name, event VastID, event genomic coordinate, exon length. Columns 5-10: RetMICs PSI in cells expressing an Empty Vector (EV), EV+scramble sequence, SRRM3+ELAVL1 KD, SRRM3+scramble sequence, SRRM4+ELAVL1 KD and SRRM4+scramble. ΔPSI between the different OE and KD conditions is provided in columns 10-13. To reliably estimate inclusion levels, only events with sufficient read coverage are shown.</p

Table 5.6

  Table 5.6: Source data for Rfx1/2/3 KO analysis. GE datasets for WT and Rfx1/2/3 KO ependymal cell. Each sheet contains Ensembl ID, gene symbol, expression levels (cRPKM) for Olfactory-Sperm dataset, spermatocytes-specific genes, olfactory-specific genes and cilia genes. </p