The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration.

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

Rapid and efficient generation of mature retinal organoids derived from human pluripotent stem cells via optimized pharmacological modulation of Sonic hedgehog, activin A, and retinoic acid signal transduction.

Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population.

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel-Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Rotenone-induced inner retinal degeneration via presynaptic activation of voltage-dependent sodium and L-type calcium channels in rats.

Rotenone, a mitochondrial complex I inhibitor, causes retinal degeneration via unknown mechanisms. To elucidate the molecular mechanisms of its action, we further characterized a rat model of rotenone-induced retinal degeneration. Intravitreal injection of rotenone (2 nmol/eye) damaged mainly the inner retinal layers, including cell loss in the ganglion cell and inner nuclear layers, which were very similar to those induced by 10 nmol/eye N-methyl-D-aspartate (NMDA). These morphological changes were accompanied by the reduced b-wave amplitude of electroretinogram, and increased immunostaining of 2,4-dinitrophenyl, an oxidative stress marker. Rotenone also downregulated expression of neurofilament light-chain gene (Nfl) as a retinal ganglion cell (RGC) marker. This effect was prevented by simultaneous injection of rotenone with antioxidants or NMDA receptor antagonists. More importantly, voltage-dependent sodium and L-type calcium channel blockers and intracellular calcium signaling modulators remarkably suppressed rotenone-induced Nfl downregulation, whereas none of these agents modified NMDA-induced Nfl downregulation. These results suggest that rotenone-induced inner retinal degeneration stems from indirect postsynaptic NMDA stimulation that is triggered by oxidative stress-mediated presynaptic intracellular calcium signaling via activation of voltage-dependent sodium and L-type calcium channels.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

The investigation of synovial genomic targets of bucillamine with microarray technique.

OBJECTIVE: To identify the molecular mechanisms of bucillamine activity, global gene expression analysis and pathway analysis were conducted using IL-1 beta-stimulated human fibroblast-like synovial cells (FLS). METHODS: Normal human FLS were treated with IL-1 beta in the presence or absence of 10 and 100 microM bucillamine for 6 h. Total RNA was extracted and global gene expression levels were detected using a 44 k human whole genome array. Data were analyzed using Ingenuity pathway analysis. RESULTS: Numerous pathways were activated by IL-1 beta stimulation. At both concentrations, bucillamine suppressed nine signal pathways stimulated by IL-1 beta. CONCLUSIONS: Bucillamine effectively inhibited fibroblast growth factor (FGF) signaling and tight junction signaling activated by IL-1 beta in FLS. Suppression of these signal pathways may correlate with the pharmacologic mechanisms of bucillamine. In particular, the suppression of FGF signaling by bucillamine is remarkable because the activation of FGF signaling may be involved in rheumatoid arthritis pathology.

Chemical proteasome inhibition as a novel animal model of inner retinal degeneration in rats.

Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.

Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model.

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

Spermidine Oxidation-Mediated Degeneration of Retinal Pigment Epithelium in Rats.

Retinal pigment epithelium (RPE) degeneration is a crucial event in dry age-related macular degeneration and gyrate atrophy. The polyamine spermidine has been shown to induce RPE cell death in vitro. The present study aimed to establish a novel in vivo model of spermidine-induced RPE degeneration and to determine whether spermidine-induced RPE cell death involves oxidative mechanisms. In this study, spermidine caused ARPE-19 cell death in a concentration-dependent manner. This effect was prevented by removal of serum from the culture medium or treatment with amine oxidase inhibitors, N-acetylcysteine (NAC), or aldehyde dehydrogenase (ALDH). Intravitreal injection of spermidine into rats significantly increased the permeability of the blood-retinal barrier and decreased the amplitudes of scotopic electroretinogram a- and b-waves. Histological analysis revealed that spermidine induced vacuolation, atrophy, and dropout of RPE cells, leading to the disruption of photoreceptor outer segments. Simultaneous intravitreal administration of NAC and ALDH with spermidine prominently inhibited the functional and morphological changes induced by spermidine. In conclusion, this study demonstrated that the intravitreal administration of spermidine induced RPE cell dysfunction and death followed by photoreceptor degeneration in rats. These effects of spermidine are thought to be mediated by oxidative stress and a toxic aldehyde generated during spermidine oxidation.

Identification of novel genes preferentially expressed in the retina using a custom human retina cDNA microarray.

PURPOSE: To construct a custom cDNA microarray for comprehensive human retinal gene expression profiling and apply it to the identification of genes that are preferentially expressed in the retina. METHODS: A cDNA microarray was constructed based on the predicted human retina gene expression profile according to expressed sequence tag (EST) databases. Gene expression profiles were obtained from five human retinas, two livers, and the cerebral cortical regions of two brains. Each sample was studied in duplicate, using a reference sample experimental design. Retina-enriched genes were identified by using the significance analysis for microarray (SAM) algorithm. Quantitative real time PCR was used to confirm microarray results. Bioinformatic analysis was performed to compare the array results with expression data available from public databases. RESULTS: The cDNA microarray contains 10,034 sequences: 67% represent known genes and 33% represent ESTs. Differential hybridization with the array identified, in addition to known retinal genes, 186 retina-enriched genes that do not have known retinal function. Of these, 96 represent novel genes. Quantitative real-time PCR of 11 of the identified genes and ESTs confirmed their retina-enriched expression pattern. Bioinformatic analysis of EST databases suggests that of the 186 genes, approximately 40% are predominantly expressed in the retina, whereas the remainder show significant expression in other tissues. Comparison of this study's microarray-based retina-enriched gene set with three published similar sets identified using complementary high-throughput approaches demonstrated only limited overlap of the identified genes. CONCLUSIONS: Because previous studies have demonstrated that many retina-enriched genes are crucial for maintaining normal retinal function, the genes identified here are likely to include ones that have important roles in the retina and ones that when mutated can cause or modulate retinal disease. In addition, the retina custom array should provide a useful resource for comparing expression profiles between normal and diseased human retinas.

Identification of gene expression changes associated with the progression of retinal degeneration in the rd1 mouse.

PURPOSE: One approach to gaining insight into the biological pathways contributing to rod and cone photoreceptor death is to identify patterns of gene expression changes. In the present study, a custom retinal microarray was developed to analyze the rd1 mouse, a well-characterized animal model of human retinal degeneration. METHODS: A microarray was constructed containing cDNA fragments corresponding to genes known or postulated to be involved in normal retinal function, development, and disease. Gene expression in rd1 retina was compared with age-matched control retinas at three time points: the peak of rod degeneration (postnatal day [P]14), early in cone degeneration (P35), and during cone degeneration (P50). Selected microarray results were confirmed with real-time PCR. The cellular distribution of one of the differentially expressed genes, dickkopf 3 (Dkk3), was assessed by in situ hybridization. RESULTS: At each stage of degeneration, there was only limited overlap of the genes that showed increased expression, suggesting the involvement of temporally distinct molecular pathways. Genes active in transport mechanisms and in signaling pathways were differentially expressed during rod degeneration, whereas genes with functions in protein modification and cellular metabolism were differentially expressed during cone degeneration. Increased expression of genes involved in cell proliferation pathways and oxidative stress was observed at each time point. CONCLUSIONS: These microarray results provide clues to understanding the molecular pathways underlying photoreceptor degeneration and indicate directions for future studies. In addition, comparisons of normal and degenerated retina identified numerous genes and ESTs that are potentially enriched in rod photoreceptors.

Comparative gene expression analysis of murine retina and brain.

PURPOSE: Several high-throughput studies have described gene expression in the central nervous system (CNS), and recently there has been increasing interest in analyzing how gene expression compares in different regions of the CNS. As the retina is often used as a model system to study CNS development and function, we compared retina and brain gene expression using microarray analyses. METHODS: Mouse retina, brain and liver RNA was hybridized to a custom cDNA microarray containing 5,376 genes and ESTs, and the data from the quantified scanned images were analyzed using Bioconductor and SAM. Preferential retina expression was confirmed by real-time PCR. The cellular distribution of genes newly identified as retina enriched genes was determined by immunohistochemistry. RESULTS: Using stringent statistical analyses we identified 733 genes that were preferentially expressed in retina and 389 in brain. The retina-liver hybridizations identified an additional 837 retina enriched genes. The cellular distribution in the retina was determined for two genes that had not previously been reported to be expressed in the retina, the transcription regulatory proteins EWS and PCPB1. Both proteins were found primarily in the inner nuclear layer. Finally, a comparison of the microarray data to publicly available SAGE and EST library databases demonstrated only limited overlap of the sets of retina enriched genes identified by the different methodologies. The preferential retinal expression of a subset of genes from the microarray, which were not identified as differentially expressed by other methods, was confirmed by quantitative PCR. CONCLUSIONS: The finding of differences in the groups of identified retina enriched genes from the various profiling techniques supports the use of multiple approaches to obtain a more complete description of retinal gene expression. Characterization of gene expression profiles of retina and brain may facilitate the understanding of the processes that underlie differences between the retina and other parts of the central nervous system.

Novel common variants and susceptible haplotype for exfoliation glaucoma specific to Asian population.

The common variants in lysyl oxidase-like 1 gene (LOXL1) are associated with exfoliation glaucoma (XFG) patients developed through exfoliation syndrome (XFS). However, the risk allele of a variant in LOXL1 has been found to be inverted between Asian and Caucasian populations. Therefore, we newly performed a genome-wide association study using 201 XFS/XFG and 697 controls in Japanese, and identified 34 genome-wide significant single-nucleotide polymorphisms (SNPs) distributing in not only LOXL1 but also TBC1D21 and PML at the 15q24.1 locus. These SNPs were confirmed by an independent population consisted of 121 XFS/XFG and 263 controls in Japanese. Moreover, further analyses revealed a unique haplotype structure only from the combination of TBC1D21 and LOXL1 variants showing a high XFS/XFG susceptibility specific for the Asian population. Although there still should be other gene(s) in the other region(s) contributing to the disease process, these results suggested that the combination of newly discovered variants in these genes might be useful for precise XFG risk assessment, as well as for elucidating the molecular mechanism of XFG pathogenesis through XFS.

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese.

BACKGROUND: To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. METHODS AND PRINCIPAL FINDINGS: We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8 × 10(-10)). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)--POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)--and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.

An approach to predict the risk of glaucoma development by integrating different attribute data.

Primary open-angle glaucoma (POAG) is one of the major causes of blindness worldwide and considered to be influenced by inherited and environmental factors. Recently, we demonstrated a genome-wide association study for the susceptibility to POAG by comparing patients and controls. In addition, the serum cytokine levels, which are affected by environmental and postnatal factors, could be also obtained in patients as well as in controls, simultaneously. Here, in order to predict the effective diagnosis of POAG, we developed an "integration approach" using different attribute data which were integrated simply with several machine learning methods and random sampling. Two data sets were prepared for this study. The one is the "training data set", which consisted of 42 POAG and 42 controls. The other is the "test data set" consisted of 73 POAG and 52 controls. We first examined for genotype and cytokine data using the training data set with general machine learning methods. After the integration approach was applied, we obtained the stable accuracy, using the support vector machine method with the radial basis function. Although our approach was based on well-known machine learning methods and a simple process, we demonstrated that the integration with two kinds of attributes, genotype and cytokines, was effective and helpful in diagnostic prediction of POAG.

New fluoroprostaglandin F(2alpha) derivatives with prostanoid FP-receptor agonistic activity as potent ocular-hypotensive agents.

To find new prostanoid FP-receptor agonists possessing potent ocular-hypotensive effects with minimal side effects, we evaluated the agonistic activities of newly synthesized prostaglandin F(2alpha) derivatives for the prostanoid FP-receptor both in vitro and in vivo. The iris constrictions induced by the derivatives and their effects on melanin content were examined using cat isolated iris sphincters and cultured B16 melanoma cells, respectively. The effects of derivative ester forms on miosis and intraocular pressure (IOP) were evaluated in cats and cynomolgus monkeys, respectively. Of these derivatives, 6 out of 12 compounds were more potent iris constrictors, with EC(50) values of 0.6 to 9.4 nM, than a carboxylic acid of latanoprost (EC(50)=13.6 nM). A carboxylic acid of latanoprost (100 microM) significantly increased the melanin content of cultured B16 melanoma cells, but some 15,15-difluoro derivatives, such as AFP-157 and AFP-172, did not. Topically applied AFP-168, AFP-169 and AFP-175 (isopropyl ester, methyl ester and ethyl ester forms, respectively, of AFP-172) induced miosis in cats more potently than latanoprost. AFP-168 (0.0005%) reduced IOP to the same extent as 0.005% latanoprost (for at least 8 h). These findings indicate that 15,15-difluoroprostaglandin F(2alpha) derivatives, especially AFP-168, have more potent prostanoid FP-receptor agonistic activities than latanoprost. Hence, AFP-168 may be worthy of further evaluation as an ocular-hypotensive agent.

Pharmacological characteristics of AFP-168 (tafluprost), a new prostanoid FP receptor agonist, as an ocular hypotensive drug.

To evaluate the pharmacological characteristics of AFP-168 (tafluprost), a new prostaglandin (PG) F(2alpha) derivative, we examined its receptor-binding affinities, intraocular pressure (IOP)-lowering effect, effects on aqueous humor dynamics, and stimulating effect on melanogenesis. The receptor-binding profile for AFP-172, a carboxylic acid of AFP-168, was determined by measuring muscle contractions in an organ bath, inhibition of platelet aggregation, and competitive binding of a radio-labelled ligand. For the IOP-measurement study, ocular normotensive and laser-induced ocular hypertensive cynomolgus monkeys were used, and IOP was measured using a pneumatonograph. For the studies of aqueous humor dynamics, IOP (Goldmann applanation tonometry), fluorophotometry, two-level constant pressure perfusion, and isotope dilution and accumulation techniques were used in ocular normotensive monkeys. The melanin contents in the medium and in the cell bodies of cultured B16-F0 melanoma cells were measured. The affinity for the FP receptor shown by AFP-172 (Ki : 0.4 nm) was 12 times that of PhXA85 ( Ki : 4.7 nm), a carboxylic acid of latanoprost. A single application of AFP-168 at 0.0025% significantly lowered IOP in both ocular normotensive and hypertensive monkeys (3.1 and 11.8 mmHg, respectively, p < 0.01) and latanoprost at 0.005% significantly lowered IOP (2.1 mmHg, p < 0.01 and 9.5 mmHg, p = 0.059 respectively). Once daily instillation of AFP-168 at 0.001, 0.0025, or 0.005% for 5 days in normotensive monkeys significantly reduced IOP not only for a few hours, but also at the drug-trough time 24hr after application. Latanoprost at 0.005% also reduced IOP, but not at the drug-trough time. AFP-168 decreased IOP mainly by increasing uveoscleral outflow by 65% (p < 0.05) and, as sometimes seen with other prostanoids, also increased total outflow facility (33% increase, p < 0.05). In cultured B16-F0 melanoma cells, AFP-172 (100 microM) did not stimulate melanogenesis, but PhXA85 (100 microM) did. These findings indicate that AFP-168 has a high affinity for the prostanoid FP receptor, has potent IOP-lowering effects in both ocular normotensive and hypertensive monkeys that exceed those of latanoprost, and has less stimulating effect on melanogenesis in melanoma cells.