The Role of MicroRNAs in Mitochondria-Mediated Eye Diseases

The retina is among the most metabolically active tissues with high-energy demands. The peculiar distribution of mitochondria in cells of retinal layers is necessary to assure the appropriate energy supply for the transmission of the light signal. Photoreceptor cells (PRs), retinal pigment epithelium (RPE), and retinal ganglion cells (RGCs) present a great concentration of mitochondria, which makes them particularly sensitive to mitochondrial dysfunction. To date, visual loss has been extensively correlated to defective mitochondrial functions. Many mitochondrial diseases (MDs) show indeed neuro-ophthalmic manifestations, including retinal and optic nerve phenotypes. Moreover, abnormal mitochondrial functions are frequently found in the most common retinal pathologies, i.e., glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR), that share clinical similarities with the hereditary primary MDs. MicroRNAs (miRNAs) are established as key regulators of several developmental, physiological, and pathological processes. Dysregulated miRNA expression profiles in retinal degeneration models and in patients underline the potentiality of miRNA modulation as a possible gene/mutation-independent strategy in retinal diseases and highlight their promising role as disease predictive or prognostic biomarkers. In this review, we will summarize the current knowledge about the participation of miRNAs in both rare and common mitochondria-mediated eye diseases. Definitely, given the involvement of miRNAs in retina pathologies and therapy as well as their use as molecular biomarkers, they represent a determining target for clinical applications

The VersaLive platform enables microfluidic mammalian cell culture for versatile applications

Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents' consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and usually require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple applications (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis, plasmid transfection) in mammalian cell lines and primary cells. VersaLive could replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research

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)

Study of the functional role of microRNAs in the regulatory networks underlying vertebrate eye development

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They exert diverse functions in controlling normal tissue and organ development and physiology. Many miRNAs show spatially and temporally restricted expression patterns during vertebrate eye development but the roles of individual miRNAs in controlling this process remain however, largely unknown. The aim of my thesis was to shed further light on the role of specific miRNAs in regulating basic processes of ocular development mainly by exploiting the medakafish (Oryzias latipes) model system. In particular, I focused my attention on the miRNA subfamily constituted by miR-181a and miR-181b, which are expressed in the Inner Nuclear Layer (INL) and Ganglion Cell Layer (GCL) of the vertebrate retina. Morpholinomediated combined knockdown of miR-181a/b function in medakafish results in a specific retinal phenotype characterized by the reduction of Inner Plexiform Layer (IPL) thickness, without any apparent reduction in the number of retinal cells. To dissect this phenotype further, I studied the consequences of miR- 181a/b ablation in two medakafish transgenic lines, namely GFP-Six3.2 and GFP-Ath5, in which GFP expression can be specifically visualized in amacrine and retinal ganglion cells (RGCs), respectively. This analysis revealed that miR-181a/b exert a role in the specification and growth of amacrine and RGC axons. The above alterations translate into an impairment of retinal circuits assembly and to visual function defects, as assessed by the evaluation of the Optokinetic Response (OKR) behavioral test. Using a combination of bioinformatic, as well as on in vitro and in vivo experimental approaches, I identified ERK2, a kinase member of the MAPK signaling cascade, as one of the direct targets of these two microRNAs. I demonstrated that the function of miR-181a/b in growth cone cytoskeleton remodeling during retinal development are largely mediated by ERK2 targeting and by the modulation of its downstream signaling cascade. Moreover I provide, for the first time, in vivo evidence of an antagonism between the TGF-b pathway and the ERK2 cascade in the regulation of retinal axon specification and growth, which is exerted via TGF-b regulation of miR- 181a/b levels. These data expand our knowledge on the role of miRNAs in eye patterning in vertebrates, and demonstrate that miR-181a/b-targeting of ERK2 and the consequent modulation of the MAPK cascade, in concert with TGF-b-action, play important roles in the signaling network that define the correct wiring and assembly of functional retina neural circuits

Sophisticated Gene Regulation for a Complex Physiological System: The Role of Non-coding RNAs in Photoreceptor Cells

Photoreceptors (PRs) are specialized neuroepithelial cells of the retina responsible for sensory transduction of light stimuli. In the highly structured vertebrate retina, PRs have a highly polarized modular structure to accommodate the demanding processes of phototransduction and the visual cycle. Because of their function, PRs are exposed to continuous cellular stress. PRs are therefore under pressure to maintain their function in defiance of constant environmental perturbation, besides being part of a highly sophisticated developmental process. All this translates into the need for tightly regulated and responsive molecular mechanisms that can reinforce transcriptional programs. It is commonly accepted that regulatory non-coding RNAs (ncRNAs), and in particular microRNAs (miRNAs), are not only involved but indeed central in conferring robustness and accuracy to developmental and physiological processes. Here we integrate recent findings on the role of regulatory ncRNAs (e.g., miRNAs, lncRNAs, circular RNAs, and antisense RNAs), and of their contribution to PR pathophysiology. We also outline the therapeutic implications of translational studies that harness ncRNAs to prevent PR degeneration and promote their survival and function

miR-181a/b control the assembly of visual circuitry by regulating retinal axon specification and growth

Connectivity and function of neuronal circuitry require the correct specification and growth of axons and dendrites. Here, we identify the microRNAs miR-181a and miR-181b as key regulators of retinal axon specification and growth. Loss of miR-181a/b in medaka fish (Oryzias latipes) failed to consolidate amacrine cell processes into axons and delayed the growth of retinal ganglion cell (RGC) axons. These alterations were accompanied by defects in visual connectivity and function. We demonstrated that miR-181a/b exert these actions through negative modulation of MAPK/ERK signaling that in turn leads to RhoA reduction and proper neuritogenesis in both amacrine cells and RGCs via local cytoskeletal rearrangement. Our results identify a new pathway for axon specification and growth unraveling a crucial role of miR-181a/b in the proper establishment of visual system connectivity and function

TGF-β signaling regulates RhoA levels via two independent and synergistic cascades.

<p><b>(a)</b> qRT-PCR analysis of erk2 transcripts in total eye RNA derived from St32 control-MOs, miR-181a/b morphants and TGF-β-treated miR-181a/b morphants. The TGF-β-mediated increase of miR-181a/b caused a rescue of miR-181a/b target transcripts, such as <i>prox1</i> and <i>erk2</i>, in miR-181a/b morphants. <b>(b, c)</b> Representative Western blotting on protein from St32 eyes (b) and corresponding quantification (c) show that administration of TGF-β to MO-miR-181a/b embryos leads to restoration of total-, phospho-Erk2 and RhoA protein levels. When MO-miR-181a/b embryos were treated with both TGF-β and the proteasomal inhibitor MG132, total- and phospho-Erk2 protein levels were still rescued, whereas RhoA levels were only partially rescued. Data are means +SEM.* P <0.05; **P <0.01; *** P <0.001 (two-way ANOVA).</p