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

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

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease

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)

A medaka model to study the the molecular basis of Microphthalmia with Linear Skin defects (MLS) syndrome

The Microphthalmia with linear skin defects (MLS) syndrome is an X- linked dominant male-lethal neuro-developmental disorder associated to mutations in the holocytochrome c-type synthetase (HCCS) transcript. Female patients display unilateral or bilateral microphthalmia and linear skin defects, additional features include central nervous system (CNS) malformation and mental retardation. HCCS codifies a mitochondrial protein that catalyzes the attachment of heme to both apocytochrome c and c1, necessary for proper functioning of the mitochondrial respiratory chain. Although mutation analysis clearly indicates a role for HCCS in the pathogenesis of this genetic condition, the molecular mechanisms underlying the developmental anomalies in the presence of HCCS dysfunction are still unknown. Previous studies demonstrated the early lethality of mouse embryonic Hccs knock-out stem cells. To overcome the problem of the possible embryonic lethality, we decided to generate an animal model for MLS syndrome in the medaka fish (Oryzia latipes) using a morpholino-based technology. Fish models (zebrafish and medaka) are considered good models to study developmental biology processes and in particular eye developmental defects. Three specific morpholinos directed against different portions of the olhccs transcript have been designed and injected and our data indicated that all morpholinos effectively downregulate the expression of the olhccs gene. The injection of the three different morpholinos resulted in a pathological phenotype, which resembles the human condition. Morphants displayed microphthalmia, coloboma, and microcephaly associated to a severe cardiac pathology. To date, this is the only animal model that recapitulates the phenotype observed in MLS syndrome. Analysis with markers for specific retinal cell types showed defects in differentiation of the ventral neural retina. Characterization of morphants revealed that hccs down-regulation results in impairment of mitochondrial functions, overproduction of reactive oxygen species (ROS) and a strong increase of apoptosis mediated by activation of the mitochondrial-dependent cell death pathway in the CNS and in the eyes. Our results clearly indicate that HCCS plays a critical role in mitochondria and imply that MLS should be considered a mitochondrial disease. It is well established that the intrinsic mitochondrial dependent apoptotic pathway rely on the formation of apoptosomes, which require the presence and/or the activity of cytochrome c, Apaf1, and caspase 9. Detailed studies of the mechanisms that underlie intrinsic apoptosis have shown that the heme group of cytochrome c is necessary for Apaf1 activation, apoptosome formation and activation of caspase 9. Interestingly, our data indicate that, in our model, the mitochondrial dependent apoptosis is triggered by caspase 9 activation and occur in a Bcl-dependent but apoptosome-independent manner suggesting that at least in some tissues the apoptosis can occur in a non-canonical way. Our data support the evidence of an apoptosome-indipendent activation of caspase 9 and suggest the possibility that this event might be tissue specific. Our study shed new light into the functional role of HCCS in the mitochondria. In addition, we provide strong evidences that mitochondrial mediated apoptotic events underlie microphtalmia providing new insights into the mechanisms of this developmental defect

Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA): An Unconventional Mitochondrial Disorder

Mitochondrial disorders, although heterogeneous, are traditionally described as conditions characterized by encephalomyopathy, hypotonia, and progressive postnatal organ failure. Here, we provide a systematic review of Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA), a rare, unconventional mitochondrial disorder which presents as a developmental disease; its main clinical features include microphthalmia with different degrees of severity, linear skin lesions, and central nervous system malformations. The molecular basis of this disorder has been elusive for several years. Mutations were eventually identified in three X-linked genes, i.e., HCCS, COX7B, and NDUFB11, which are all endowed with defined roles in the mitochondrial respiratory chain. A peculiar feature of this condition is its inheritance pattern: X-linked dominant male-lethal. Only female or XX male individuals can be observed, implying that nullisomy for these genes is incompatible with normal embryonic development in mammals. All three genes undergo X-inactivation that, according to our hypothesis, may contribute to the extreme variable expressivity observed in this condition. We propose that mitochondrial dysfunction should be considered as an underlying cause in developmental disorders. Moreover, LSDMCA should be taken into consideration by clinicians when dealing with patients with microphthalmia with or without associated skin phenotypes

The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes

Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non-canonical mitochondrial-dependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X-linked developmental disorder caused by mutations in the holo-cytochrome c-type synthase (HCCS) gene. By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes. We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non-canonical start-up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders. The X-linked disorder microphtalmia with skin lesions (MLS) is caused by HCCS gene mutations. The disease phenotype is now be explained by the increased non-conventional caspase-9 -mediated cell death in the brain and eyes due to HCCS impairment. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.Italian Telethon Foundation (BF, grant TGM11CB3); the Spanish MICINN (BFU2010-16031); CIBERER (PB); EMBO short-term fellowship; Company of Biologists LtdPeer Reviewe

The HOPS Complex Subunit VPS39 controls ciliogenesis through autophagy

Primary cilia are microtubule-based organelles that assemble and protrude from the surface of most mammalian cells during quiescence. The biomedical relevance of cilia is indicated by disorders ascribed to cilia dysfunction, known as ciliopathies, that display distinctive features including renal cystic disease. In this report, we demonstrate that VPS39, a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, acts as a negative regulator of ciliogenesis in human renal cells, by controlling the localization of the intraflagellar transport 20 (IFT20) protein at the base of cilia through autophagy. Moreover, we show that VPS39 controls ciliogenesis through autophagy also in vivo in renal tubules of Medaka fish. These observations suggest a direct involvement of the HOPS complex in the regulation of autophagy-mediated ciliogenesis and eventually in target selection. Interestingly, we show that the impact of autophagy modulation on ciliogenesis is cell-type dependent and strictly related to environmental stimuli. This report adds a further tile to the cilia-autophagy connection and suggests that VPS39 could represent a new biological target for the recovery of the cilia-related phenotypes observed in the kidneys of patients affected by ciliopathies