Serum miRNA modulations indicate changes in retinal morphology.

Background: Age-related macular degeneration (AMD) is the leading cause of vision loss in the developed world and the detection of its onset and progression are based on retinal morphological assessments. MicroRNA (miRNA) have been explored extensively as biomarkers for a range of neurological diseases including AMD, however differences in experimental design and the complexity of human biology have resulted in little overlap between studies. Using preclinical animal models and clinical samples, this study employs a novel approach to determine a serum signature of AMD progression. Methods: Serum miRNAs were extracted from mice exposed to photo-oxidative damage (PD; 0, 1, 3 and 5 days), and clinical samples from patients diagnosed with reticular pseudodrusen or atrophic AMD. The expression of ~800 miRNAs was measured using OpenArray™, and differential abundance from controls was determined using the HTqPCR R package followed by pathway analysis with DAVID. MiRNA expression changes were compared against quantifiable retinal histological indicators. Finally, the overlap of miRNA changes observed in the mouse model and human patient samples was investigated. Results: Differential miRNA abundance was identified at all PD time-points and in clinical samples. Importantly, these were associated with inflammatory pathways and histological changes in the retina. Further, we were able to align findings in the mouse serum to those of clinical patients. Conclusion: In conclusion, serum miRNAs are a valid tool as diagnostics for the early detection of retinal degeneration, as they reflect key changes in retinal health. The combination of pre-clinical animal models and human patient samples led to the identification of a preliminary serum miRNA signature for AMD. This study is an important platform for the future development of a diagnostic serum miRNA panel for the early detection of retinal degeneration.

Hallmarks of lens aging and cataractogenesis.

Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.

MicroRNA-223 Regulates Retinal Function and Inflammation in the Healthy and Degenerating Retina.

INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding RNA molecules that have powerful regulatory properties, with the ability to regulate multiple messenger RNAs (mRNAs) and biological pathways. MicroRNA-223-3p (miR-223) is known to be a critical regulator of the innate immune response, and its dysregulation is thought to play a role in inflammatory disease progression. Despite miR-223 upregulation in numerous neurodegenerative conditions, largely in cells of the myeloid lineage, the role of miR-223 in the retina is relatively unexplored. Here, we investigated miR-223 in the healthy retina and in response to retinal degeneration. METHODS: miR-223-null mice were investigated in control and photo-oxidative damage-induced degeneration conditions. Encapsulated miR-223 mimics were intravitreally and intravenously injected into C57BL/6J wild-type mice. Retinal functional responses were measured using electroretinography (ERG), while extracted retinas were investigated by retinal histology (TUNEL and immunohistochemistry) and molecular analysis (qPCR and FACS). RESULTS: Retinal function in miR-223-/- mice was adversely affected, indicating that miR-223 may be critical in regulating the retinal response. In degeneration, miR-223 was elevated in the retina, circulating serum, and retinal extracellular vesicles. Conversely, retinal microglia and macrophages displayed a downregulation of miR-223. Further, isolated CD11b+ inflammatory cells from the retinas and circulation of miR-223-null mice showed an upregulation of pro-inflammatory genes that are critically linked to retinal inflammation and progressive photoreceptor loss. Finally, both local and systemic delivery of miR-223 mimics improved retinal function in mice undergoing retinal degeneration. CONCLUSION: miR-223 is required for maintaining normal retinal function, as well as regulating inflammation in microglia and macrophages. Further investigations are required to determine the targets of miR-223 and their key biological pathways and interactions that are relevant to retinal diseases. Future studies should investigate whether sustained delivery of miR-223 into the retina is sufficient to target these pathways and protect the retina from progressive degeneration.

A novel NADPH oxidase inhibitor targeting Nox4 in TGFß-induced lens epithelial to mesenchymal transition.

Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGFß-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3  µM of GLX7013114, and then treated with 200 pg/ml of TGF-ß2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGFß-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGFß-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGFß and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGFß-induced gene expression of α-smooth muscle actin (αSMA), collagen 1a and fibronectin. GLX7013114, given that it appears to block aspects of TGFß-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies.

Postnatal Development of Spasticity Following Transgene Insertion in the Mouse ßIV Spectrin Gene (SPTBN4).

BACKGROUND: The L25 mouse line was generated by random genomic insertion of a lens-specific transgene. Inbreeding of L25 hemizygotes revealed an unanticipated spastic phenotype in the hind limbs. OBJECTIVE: The goals were to characterize the motor phenotype in the L25 mice and to map the transgene insert site within the mouse genome. METHODS: Six pairs of L25+/- mice were repeatedly mated. Beginning at weaning, all progeny were inspected for body weight and motor signs twice weekly until they displayed predefined ethical criteria for termination. The transgene insert site was determined by whole genome sequencing. Western blotting was used to compare the expression levels of beta-IV spectrin protein in the brain. RESULTS: Matings of hemizygous L25+/- × L25+/- mice yielded 20% (29/148) affected weanlings, identified by an abnormal retraction of the hind limbs when lifted by the tail, and a fine tremor. Affected mice were less mobile and grew more slowly than wild-type littermates. All affected mice required termination due to >15% loss of body weight (50% survival age 92 days). At the endpoint, mice showed varying degrees of spastic paresis or spastic paralysis localised to the hind limbs. Motor endplates remained fully innervated. Genome sequencing confirmed that the transgene was inserted in the locus of ßIV spectrin of L25 mice. Western blotting indicated that this random insertion had greatly reduced the expression of ßIV spectrin protein in the affected L25 mice. CONCLUSIONS: The results confirm the importance of ßIV spectrin for maintaining central motor pathway control of the hind limbs, and provide a developmental time course for the phenotype.

Nox4 Plays a Role in TGF-ß-Dependent Lens Epithelial to Mesenchymal Transition.

PURPOSE: Transforming growth factor-ß induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-ß-driven EMT is dependent on the expression of the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-ß-induced lens EMT and determine whether it is required for this pathologic process. METHODS: Rat lens epithelial explants were used to investigate the role of Nox4 in TGF-ß-driven lens EMT. Nox1-4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH-oxidase-produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-ß-driven EMT. RESULTS: We demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-ß treatment induces Nox4 (but not Nox1-3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-ß treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-ß treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression. CONCLUSIONS: Transforming growth factor-ß drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.