Ultrastructural Changes and Expression of PCNA and RPE65 in Sodium Iodate-Induced Acute Retinal Pigment Epithelium Degeneration Model.

Alteration in retinal pigment epithelium (RPE) results in the visual dysfunction and blindness of retinal degenerative diseases. Injection of sodium iodate (NaIO3) generates degeneration of RPE. We analyzed the sequential ultrastructure and expression of proliferating cell nuclear antigen (PCNA) and retina-specific RPE65 in NaIO3-induced retinal degeneration model. Adult male rats were injected 1% NaIO3 (50 mg/kg) and eyes were enucleated at 1, 3, 5, 7 and 14 days post-injection (DPI), fixed, and processed for histological analysis. NaIO3-induced retinal degeneration was successfully established. At 1 DPI, most RPE cells were degenerated and replaced by a few proliferating RPE cells in the peripheral area. At 3 DPI, the RPE and photoreceptor out segments (POS) underwent a marked morphological change, including POS disruption, accumulation of residual bodies in RPE and POS, and hyperplasia of the RPE cell. At 5 DPI, POS showed a maximum increase in the outer segment debris and the retina showed partial detachment. These abnormal morphological changes gradually decreased by day 7. At 14 DPI, the damaged RPE and POS were partially regenerated from the peripheral to the central region. Expression of PCNA and RPE65 increased from day 3 onward. The damaged RPE showed earlier expression of PCNA and RPE65 than POS. The RPE damaged by NaIO3 rapidly proliferated to put down roots on Bruch's membrane from the peripheral retina and proliferation and hyperplasia of the RPE had a regular direction of progress. Therefore, NaIO3-induced acute changes in retina mimic the patho-morphologic features of RPE-related diseases.

Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells.

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. A microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

Fundus Camera-Delivered Light-Induced Retinal Degeneration in Mice With the RPE65 Leu450Met Variant is Associated With Oxidative Stress and Apoptosis.

PURPOSE: Oxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD. METHODS: The retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed. RESULTS: Both protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition. CONCLUSIONS: We characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

miR-410 Inhibition Induces RPE Differentiation of Amniotic Epithelial Stem Cells via Overexpression of OTX2 and RPE65.

The retinal pigment epithelium (RPE) is a highly specialized cell type located between the choroid and neural retina of the eye. RPE degeneration causes irreversible visual impairment, extending to blindness. Cell therapy has recently emerged as a potential therapeutic approach for retinal degeneration. MicroRNA-based differentiation of stem cells is a new strategy for producing tissue-specific cell types. In this study, we developed a novel microRNA-based strategy for RPE induction from human amniotic epithelial stem cells (AESCs). We identified microRNAs involved in RPE development in AESCs. Of 29 putative human RPE-relevant microRNAs, microRNA-410 (miR-410) was predicted to target multiple RPE development-relevant genes. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including OTX2, RPE65, Bestrophin and EMMPRIN. These RPE-like cells were morphologically altered toward a cobblestone-like shape and were able to phagocytize microbeads. We showed that miR-410 directly regulates predicted target genes OTX2 and RPE65. Our microRNA-based strategy demonstrated RPE differentiation in AESCs by treatment of an antisense microRNA-410 (anti-miR-410), without the use of additional factors or exogenous transduction. These findings suggest that miR-410 inhibition can be a useful tool for directed cell differentiation and an attractive method for cell therapy in human retinal degenerative diseases.

The newt reprograms mature RPE cells into a unique multipotent state for retinal regeneration.

The reprogramming of retinal pigment epithelium (RPE) cells in the adult newt immediately after retinal injury is an area of active research for the study of retinal disorders and regeneration. We demonstrate here that unlike embryonic/larval retinal regeneration, adult newt RPE cells are not directly reprogrammed into retinal stem/progenitor cells; instead, they are programmed into a unique state of multipotency that is similar to the early optic vesicle (embryo) but preserves certain adult characteristics. These cells then differentiate into two populations from which the prospective-neural retina and -RPE layers are formed with the correct polarity. Furthermore, our findings provide insight into the similarity between these unique multipotent cells in newts and those implicated in retinal disorders, such as proliferative vitreoretinopathy, in humans. These findings provide a foundation for biomedical approaches that aim to induce retinal self-regeneration for the treatment of RPE-mediated retinal disorders.

The Rpe65 rd12 allele exerts a semidominant negative effect on vision in mice.

PURPOSE: The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself. METHODS: A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. RESULTS: The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes. CONCLUSIONS: The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.

Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.

RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice.

PURPOSE: Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice. METHODS: Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina. RESULTS: High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice. CONCLUSIONS: RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

Cis-trans isomerase gene in psychrophilic Pseudomonas syringae is constitutively expressed during growth and under conditions of temperature and solvent stress.

In a recent study, we established that psychrophilic Pseudomonas syringae (Lz4W) requires trans-monounsaturated fatty acid for growth at higher temperatures (Kiran et al. in Extremophiles, 2004). It was also demonstrated that the cti gene was highly conserved and exhibited high sequence identity with cti of other Pseudomonas spp. (Kiran et al. in Extremophiles, 2004). Therefore it would be interesting to understand the expression of the cti gene so as to unravel the molecular basis of adaptation of microorganisms to high temperature. In the present study, the expression of cti was monitored by RT-PCR analysis during different growth stages and under conditions of high temperature and solvent stress in P. syringae. Results indicated that the cti gene is constitutively expressed during different stages of growth and the transcript level is unaltered even under conditions of temperature and solvent stress implying that the observed increase in trans-monounsaturated fatty acids (Kiran et al. in Extremophiles, 2004) is not under transcriptional control. A putative promoter present in the intergenic region of the metH and cti gene has also been characterized. The translation start site ATG, the Shine-Dalgarno sequence AGGA and the transcription start site "C" were also identified. These results provide evidence for the first time that the cti gene is constitutively expressed under normal conditions of growth and under conditions of temperature and solvent stress thus implying that the Cti enzyme is post-transcriptionally regulated.

Comparative tyrosine degradation in Vibrio cholerae strains. The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell.

The relationship between L-tyrosine catabolism and melanin formation was studied in the Vibrio cholerae strains ATCC 14035 and CECT 557. It is shown that both strains degrade L-tyrosine by the same pathway as eukaryotic cells, giving homogentisate as intermediate. ATCC 14035, an O1 strain, which is not able to grow using L-tyrosine as sole carbon and energy source, but it forms pyomelanin from homogentisate. The second strain, which is non-O1, is able to grow using L-tyrosine as sole carbon and energy source, but it does not form any pigment. Both strains contain all the enzymes involved in the L-tyrosine catabolism. The three late enzymes of the pathway, homogentisate oxygenase, maleylacetoacetate isomerase and fumarylacetoacetate hydrolase, are induced by L-tyrosine, but the degree of induction is much lower in the ATCC 14035 strain. Thus, the distal part of the pathway becomes the rate-limiting steps in the L-tyrosine catabolism, explaining homogentisate accumulation and pyomelanogenesis in this strain. It is proposed that V. cholerae might be a useful prokaryotic model to show that alkaptonuria and other diseases related to L-tyrosine metabolism could occur in animals even when no particular enzyme involved in that pathway is lacking.