The relationship between equol production status and normal tension glaucoma.

PURPOSE: Equol is metabolized by intestinal bacteria from soy isoflavones and is chemically similar to estrogen. Dietary habits, such as consumption of soy products, influence equol production. A relationship between glaucoma and estrogen has been identified; here, we investigated the relationship between equol production status and glaucoma in Japan. METHODS: We recruited 68 normal-tension glaucoma (NTG) patients (male to female ratio 26:42, average age 63.0 ± 7.6 years) and 31 controls (male to female ratio 13:18, average age 66.0 ± 6.3 years) from our hospital. All women included were postmenopausal. Urinary equol concentration was quantified with the ELISA method. MD was calculated based on the Humphrey visual field. The association between MD and equol was analyzed with Spearman's rank correlation coefficient. The Mann-Whitney U test was used to compare the equol-producing (> 1 µM) and non-producing (< 1 µM) subjects. We also investigated the association between equol and glaucoma with a logistic regression analysis. RESULTS: There was a significant association between equol and MD (r = 0.36, P < 0.01) in the NTG patients. Glaucoma, represented by MD, was significantly milder in the equol-producing subjects than the non-equol producing subjects (P = 0.03). A multivariate analysis revealed the independent contributions of equol, cpRNFLT, and IOP to MD (P = 0.03, P = 0.04, and P < 0.01, respectively). CONCLUSION: Our results suggest that equol, acting through estrogen receptor-mediated neuroprotective effects, might be involved in suppressing the progression of NTG. This result also adds to evidence that glaucoma may be influenced by lifestyle.

MicroRNA profiles in aqueous humor between pseudoexfoliation glaucoma and normal tension glaucoma patients in a Korean population.

We aimed to obtain microRNA (miRNA) profiles of patients with pseudoexfoliation (PEX) glaucoma or normal-tension glaucoma (NTG) compared to normal controls using individual aqueous humor (AH) samples and investigate the role of miRNAs in the pathogenesis of PEX glaucoma compared to NTG in Korean. AH (80-120 µl) was collected before cataract surgery or trabeculectomy from 26 Korean subjects (eleven with PEX glaucoma, age-matched eight NTG, and seven controls). RNA sequencing was conducted for RNA samples extracted from 26 AH samples. Bioinformatics analysis was performed for targets and related pathways. A total of 334 and 291 discrete miRNAs were detected in AH samples of PEX glaucoma and NTG patients, respectively. Two significantly upregulated miRNAs (hsa-miR-30d-5p and hsa-miR-320a) and ten significantly downregulated miRNAs (hsa-miR-3156-5p, hsa-miR-4458, hsa-miR-6717-5p, hsa-miR-6728-5p, hsa-miR-6834-5p, hsa-miR-6864-5p, hsa-miR-6879-5p, hsa-miR-877-3p, hsa-miR-548e-3p, and hsa-miR-6777-5p) in PEX glaucoma patients compared to control (fold-change > 2, p < 0.05) were found. In NTG patients, ten significantly upregulated and two downregulated miRNAs compared to control were found. Only hsa-miR-6777-5p was commonly downregulated in both PEX glaucoma and NTG patients. Related pathways were proteoglycans in cancer, glioma, and TGF-beta signaling pathway in PEX glaucoma. These differentially expressed miRNAs between PEX glaucoma and NTG samples suggest the possible role of miRNA in the pathogenesis of glaucoma, further implying that pathogenic mechanisms may differ between different types of glaucoma.

Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma.

Previous reports have shown possible association between altered protein levels in aqueous humor (AH) and normal-tension glaucoma (NTG), but the underlying pathogenetic mechanism as well as specific molecular biomarkers for NTG remains still elusive. Here, we aimed to identify novel biomarkers for advanced NTG by analyzing the proteome of patient-derived AH and their correlation with various functional and structural parameters from the visual field test (VF), optical coherence tomography (OCT), and OCT angiography (OCTA). We determined differentially expressed proteins (DEPs) of the AH of patients with advanced NTG (n = 20) using label-free quantitative (LFQ) proteomics with pooled samples and data-independent acquisition (DIA) analysis with individual samples, and the roles of AH DEPs in biological pathways were evaluated using bioinformatics. We identified 603 proteins in the AH of patients with advanced NTG, and 61 of them were selected as DEPs via global proteome LFQ profiling. Individual DIA analyses identified a total of 12 DEPs as biomarker candidates, seven of which were upregulated, and five were downregulated. Gene ontology enrichment analysis revealed that those DEPs were mainly involved in the immune response. Moreover, IGFBP2, ENO1, C7, B2M, AMBP, DSP, and DCD showed a significant correlation with the mean deviation of VF and with peripapillary and macular parameters from OCT and OCTA. The present study provides possible molecular biomarkers for the diagnosis of advanced NTG.

Neuroprotective effects of bone marrow Sca-1+ cells against age-related retinal degeneration in OPTN E50K mice.

Glaucoma is characterized by retinal ganglion cell (RGC) death, the underlying mechanisms of which are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal-tension glaucoma (NTG), which directly affects RGCs in the absence of high intraocular pressure and causes severe glaucomatous symptoms in patients. Bone marrow (BM) stem cells have been demonstrated to play a key role in regenerating damaged tissue during ageing and disease through their trophic effects and homing capability. Here, we separated BM stem cells into Sca-1+ and Sca-1- cells and transplanted them into lethally irradiated aged OPTN E50K mice to generate Sca-1+ and Sca-1- chimaeras, respectively. After 3 months of BM repopulation, we investigated whether Sca-1+ cells maximized the regenerative effects in the retinas of NTG model mice with the OPTN E50K mutation. We found that the OPTN E50K mutation aggravated age-related deficiency of neurotrophic factors in both retinas and BM during NTG development, leading to retinal degeneration and BM dysfunction. Sca-1+ cells from young healthy mice had greater paracrine trophic effects than Sca-1- cells and Sca-1+ cells from young OPTN E50K mice. In addition, Sca-1+ chimaeras demonstrated better visual functions than Sca-1- chimaeras and untreated OPTN E50K mice. More Sca-1+ cells than Sca-1- cells were recruited to repair damaged retinas and reverse visual impairment in NTG resulting from high expression levels of neurotrophic factors. These findings indicated that the Sca-1+ cells from young, healthy mice may have exhibited an enhanced ability to repair retinal degeneration in NTG because of their excellent neurotrophic capability.

Proteomic analysis of aged and OPTN E50K retina in the development of normal tension glaucoma.

Progressive degeneration of retinal ganglion cells (RGCs) is a major characteristic of glaucoma, whose underlying mechanisms are still largely unknown. An E50K mutation in the Optineurin (OPTN) gene is a leading cause of normal tension glaucoma (NTG), directly affecting RGCs without high intraocular pressure and causing severe glaucomatous symptoms in clinical settings. A systematic analysis of the NTG mouse model is crucial for better understanding of the underlying pathological mechanisms for glaucoma. To elucidate proteomic and biochemical pathway alterations during NTG development, we established an OPTN E50K mutant mouse model through CRISPR/Cas9. Retinal proteins from resulting mice exhibiting glaucomatous phenotypes were subject to tandem mass tag-labeled quantitative proteomics and then analyzed through bioinformatics methods to characterize the molecular and functional signatures of NTG. We identified 6364 quantitative proteins in our proteomic analysis. Bioinformatics analysis revealed that OPTN E50K mice experienced protein synthesis dysregulation, age-dependent energy defects and autophagy-lysosome pathway dysfunction. Certain biological features, including amyloid deposition, RNA splicing, microglia activation and reduction of crystallin production, were similar to Alzheimer's disease. Our study is the first to describe proteomic and biochemical pathway alterations in NTG pathogenesis during disease advancement. Several proteomic signatures overlapped with retinal changes found in the ad mice model, suggesting the presence of common mechanisms between age-related degenerative disorders, as well as prospective new targets for diagnostic and therapeutic strategies.

Bioinorganic chemistry of open-angle glaucoma: A review.

At present, physical methods of chemical analysis are constantly improving providing large amount of data on elemental composition of organs and tissues. However, only few works describe the correlation (or the potential connection) between the general or local bioelemental imbalances and specific biochemical reactions that are involved in pathogenesis of certain diseases. This review describes primary open-angle glaucoma (POAG) - one of the most common ophthalmic diseases - in terms of elemental chemistry. The authors look into the impact that various subgroups of elements have on passive and active processes of homeostasis regulation and hydrodynamic balance in the eye. Alkaline metals and their analogues (K, Na, Li, Rb, Cs) influence hydrostatics and hydrodynamics by means of both K-Na pumps and osmosis. Alkaline-earth elements and their analogues (Ca, Mg, Sr, Ba, Be) are involved in biomineralization and intercellular interaction in the drainage areas. Chalcophile metals and their analogues (Zn, Cu, Hg, Co, Ni, Cd, Pb, Mo, Sb) regulate redox reactions. They are the cofactors of enzymes that support structural homeostasis of the drainage area. Siderophile metals (Fe, Mn, Cr, Rh) regulate oxidation-reduction reactions, including those associated with limited nutrition of tissues in glaucoma. The role of amphoteric metals and nonmetals (Al, Si, Ga, V, TI, Sn, Ge, Zr, W) in POAG has not been described properly, but they were noted to participate in mineralization. Structure-forming non-metals and their analogues (N, S, Se, As) are directly involved in the formation of protein and non-protein aggregates that prevent aqueous humor outflow. The specific role of phosphorus in the pathogenesis of glaucoma has not been described previously. The authors analyze the involvement of phosphorus in energy-dependent processes of cellular activity, which are aimed at the reprocessing of aggregates that cause aqueous humor retention.

FOXC1 variant in a family with anterior segment dysgenesis and normal-tension glaucoma.

Our study describes the glaucoma phenotype in a family with Axenfeld-Rieger syndrome (ARS) and a FOXC1 variant. Included were 20 subjects from a large three generation family of Jewish Indian ancestry. Subjects underwent a comprehensive ophthalmic examination including automated perimetry and optical coherence tomography. Eight subjects were available for molecular analysis which included whole genome sequencing on selected patients and Sanger sequencing for variant screening. Eleven patients demonstrated a wide spectrum of Axenfeld-Rieger anomaly signs and symptoms. These ranged from subtle angle abnormalities to remarkable anterior segment abnormalities such as corectopia, iris adhesions and strands. Among them, six had glaucoma and two were glaucoma suspects. Of the six subjects with glaucoma three had high-tension glaucoma and two had normal-tension glaucoma. Molecular analysis revealed a previously described pathogenic variant in the FOXC1 gene (c.378C > G p.I126M; rs104893958), in six affected patients which was not identified in two healthy siblings. Molecular analysis also revealed a PITX2 missense variant (c.28T > A p.L10M; rs755864040) which did not segregate with clinical findings and was considered likely benign. In conclusion, patients with ARS due to FOXC1 variants may present with glaucomatous optic nerve damage without apparent elevation in IOP. Normal-tension glaucoma is less commonly reported in individuals with ARS and a comprehensive glaucoma assessment may be warranted in these individuals even with normal IOP. These findings raise the possibility that glaucomatous damage associated with FOXC1 is not only due to high IOP.

EAAT1 variants associated with glaucoma.

Glaucoma is one of the leading causes of blindness characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. We reported that glutamate/aspartate transporter (GLAST) knockout mice showed progressive RGC loss and optic nerve degeneration that are similar to glaucoma. To explore the possibility that rare variants in the EAAT1 gene (the human homolog of GLAST) cause susceptibility to glaucoma, we performed targeted sequencing of EAAT1 in 440 patients with glaucoma and 450 control subjects. We identified 8 rare variants in 20 out of 440 patients, including 4 synonymous and 4 missense variants located at protein coding regions. One of these rare variants (rs117295512) showed significant association with the risk of glaucoma (OR = 10.44, P = 0.005). Furthermore, the allele frequency for loss-of-function EAAT1 variants, pAla169Gly and pAla329Thr, was 5.5 folds higher in the glaucoma (1.1%) compared with the control cohort (0.2%). These findings suggest that these rare variants may contribute to the pathogenesis of glaucoma and that loss-of-function variants in EAAT1 are present in a small number of patients with glaucoma.

Altered information flow and microstructure abnormalities of visual cortex in normal-tension glaucoma: Evidence from resting-state fMRI and DKI.

Normal tension glaucoma (NTG) is a neurodegenerative disease involves multiple brain areas, but the mechanism remains unclear. The aim of this study is to investigate the correlation between structural injury and functional reorganization in the brain of NTG, using resting-state functional MRI and diffusion kurtosis imaging (DKI) data acquired for 26 NTG patients and 24 control subjects. Granger causality analysis (GCA) was used to calculate the effective connectivity (EC) between visual cortices and the whole brain to reflect the information flow. The fractional anisotropy (FA), mean kurtosis (MK), axial kurtosis (AK), and radial kurtosis (RK) derived from DKI of visual cortices were extracted to evaluate structural injury. Microstructural abnormalities were detected in bilateral BA17, BA18, and BA19. NTG patients showed significantly decreased EC from BA17 to higher visual cortices and increase EC from higher visual cortices to BA17. The EC from BA17 to posterior cingulate cortex (PCC) and from PCC to BA17 both significantly increased, while the EC from right BA18 and BA19 to PCC significantly decreased. Decreased EC between somatosensory cortex and BA17, as well as the decreased ECs between supramarginal gyrus (SMA) and BA17/BA19 were detected. Several abnormal ECs were significantly correlated with microstructural injuries of BA17 and BA18. In conclusion, NTG causes reorganization of information flows among visual cortices and other brain areas, which is consistent with brain microstructural injury.

Retinal ganglion cell loss in kinesin-1 cargo Alcadein α deficient mice.

Maintenance of retinal ganglion cells (RGCs) activity is relied on axonal transport conveying materials required for their survival such as neurotrophic factors. Kinesin-1 undergoes anterograde transport in axons, and Alcadein α (Alcα; also called calsyntenin-1) is a major cargo adaptor protein that can drive kinesin-1 to transport vesicles containing Alcα. The long-term effects of Alcα-deficiency on retinal morphology and survival of RGCs during postnatal development were examined in Alcα knockout mice. At 1.5, 3, 6, and 15 months postnatal, the number of retrogradely labeled RGCs was determined in flat-mounted retinas of Alcα-deficient and wild-type mice. Retinal damage was assessed histologically by determining the retinal thickness. Intraocular pressure (IOP) was measured with a Tonolab tonometer. At 1.5 months postnatal, the number of retrogradely labeled RGCs was not different between wild-type and Alcα-deficient mice. However, at 3, 6, and 15 months postnatal, the number of RGCs was significantly lower in Alcα deficient mice than those of wild-type mice (143 ± 41.1 cells/mm2 vs. 208 ± 28.4 cells/mm2, respectively, at 3 months; P < 0.01). No differences were seen in retinal thickness or IOP between the two types of mice at any postnatal age. Alcα-deficient mice showed spontaneous loss of RGCs but no elevation in IOP. These mice mimic normal-tension glaucoma and will be useful for investigating the mechanism of neurodegeneration in this disorder and for developing treatments for RGC loss that does not involve changes in IOP.

Lack of Correlation between ASB10 and Normal-tension Glaucoma in a Population from the Republic of Korea.

Purpose: Ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) was identified as a novel gene for glaucoma. Since then, there have been reports on the association of ASB10 with glaucoma in various ethnic populations. In these studies, patients with different glaucoma types were included. Thus, we investigated the relationship between ASB10 and NTG in a Korean cohort.Methods: Whole-exome sequencing was performed to identify the ASB10 variants in one patient with a strong NTG family history. A total of 263 participants, comprising 157 NTG patients and 106 control subjects, were analyzed for ASB10 gene single nucleotide polymorphisms (SNPs).Results: Nine variants of the ASB10 gene were identified using whole-exome sequencing analysis, including four exonic SNPs. Of the exonic variants, three were known polymorphisms (rs3800791, rs2253592, and rs77615410), and one was newly reported (rs552803353). A nonsynonymous variant, rs552803353 was predicted as functionally damaging using PolyPhen-2. The exonic SNPs were compared against gene sequences of the control group in the NTG cohort. However, the minor allele frequency (MAF) of rs552803353 was found to be 0.029 and 0.038 in NTG cases and control subjects, respectively. The MAF of rs3800791 was found to be 0.096 and 0.118 in NTG cases and control subjects, respectively, and the MAF of rs77615410 was found to be 0.220 and 0.245 in NTG cases and control subjects, respectively, which were higher than those reported by previous studies. Genetic association analysis of four ASB10 SNPs revealed no significant difference in genotype distribution between NTG cases and control subjects in allelic, dominant, or recessive models (all, P > .05).Conclusions: The present study indicated that the MAFs of ASB10 gene polymorphisms showed a large difference among various ethnic groups, and that ASB10 gene polymorphisms may not be associated with genetic susceptibility to NTG in a Korean cohort.

Melanopsin-mediated pupillary light reflex and sleep quality in patients with normal tension glaucoma.

PURPOSE: The intrinsically photosensitive retinal ganglion cells (ipRGCs) and sleep quality are impaired in patients with primary open-angle glaucoma (POAG). In this study, we investigated whether ipRGCs and sleep quality were also impaired in patients with normal tension glaucoma (NTG). METHODS: We performed pupillometry and sleep quality assessment in 15 patients with NTG and 17 healthy age-matched controls. Pupillometry protocol consisted of monocular stimulation with high illuminance (100 lux) red (633 nm, 300 cd/m2 or 15.23 log quanta/cm2 /s) and blue light (463 nm, 332 cd/m2 or 15.27 log quanta/cm2 /s) and binocular pupil measurements. Prior to light stimulation, patients were dark-adapted for 5 min. The late postillumination pupillary response (PIPRL ate ) to blue light was used as marker of ipRGC activity. Sleep quality was assessed by Pittsburgh Sleep Quality Index (PSQI) questionnaire. RESULTS: The PIPRL ate to blue light was significantly reduced in patients with NTG compared to healthy subjects (p < 0.001), indicating impairment of the melanopsin-mediated pupillary pathway. There was no significant difference in the response elicited by red light (p = 0.6). Baseline pupil diameter and pupillary constriction amplitude to both red and blue light were reduced in patients with NTG (p < 0.05). The global score in PSQI was not significantly different between healthy controls and patients with NTG, indicating normal sleep quality (p = 0.6). Furthermore, we found no correlation between sleep parameters and pupillary light reflex parameters. CONCLUSION: Patients with NTG exhibited reduced ipRGC activity compared to healthy subjects, while no differences were observed in sleep quality.

Normal tension glaucoma-like degeneration of the visual system in aged marmosets.

The common marmoset (Callithrix jacchus) is a non-human primate that provides valuable models for neuroscience and aging research due to its anatomical similarities to humans and relatively short lifespan. This study was carried out to examine whether aged marmosets develop glaucoma, as seen in humans. We found that 11% of the aged marmosets presented with glaucoma-like characteristics; this incident rate is very similar to that in humans. Magnetic resonance imaging showed a significant volume loss in the visual cortex, and histological analyses confirmed the degeneration of the lateral geniculate nuclei and visual cortex in the affected marmosets. These marmosets did not have elevated intraocular pressure, but showed an increased oxidative stress level, low cerebrospinal fluid (CSF) pressure, and low brain-derived neurotrophic factor (BDNF) and TrkB expression in the retina, optic nerve head and CSF. Our findings suggest that marmosets have potential to provide useful information for the research of eye and the visual system.

Survival of Alpha and Intrinsically Photosensitive Retinal Ganglion Cells in NMDA-Induced Neurotoxicity and a Mouse Model of Normal Tension Glaucoma.

Purpose: We assess if α retinal ganglion cells (αRGCs) and intrinsically photosensitive retinal ganglion cells (ipRGCs) survive in mouse models of glaucoma. Methods: Two microliters of N-methyl-D-aspartate (NMDA; 1 mM) or PBS were injected intraocularly 7 days before sacrifice. Immunohistochemical analyses of the retina were performed using antibodies against RNA-binding protein with multiple splicing (RBPMS), osteopontin, and melanopsin. Immunohistochemical analyses also were performed in adult mice with glutamate/aspartate transporter (GLAST) deletion (GLAST knockout [KO] mice), a mouse model of normal tension glaucoma. Results: NMDA-induced loss of RBPMS-positive total RGCs was 58.4% ± 0.4% compared to PBS-treated controls, whereas the loss of osteopontin-positive αRGCs was 5.0% ± 0.6% and that of melanopsin-positive ipRGCs was 7.6% ± 1.6%. In GLAST KO mice, the loss of total RGCs was 48.4% ± 0.9% compared to wild-type mice, whereas the loss of αRGCs and ipRGCs was 3.9% ± 0.4% and 9.3% ± 0.5%, respectively. The distribution of survived total RGCs, αRGCs, and ipRGCs was similar regardless of the location of the retina. Conclusions: These results suggest that αRGC and ipRGC are highly tolerant to NMDA-induced neurotoxicity and NTG-like neurodegeneration in GLAST KO mice.

Association of HK2 and NCK2 with normal-tension glaucoma in a population from the Republic of Korea.

BACKGROUND: Previous studies have reported the association of HK2 and NCK2 genes with normal-tension glaucoma (NTG) in Japan, but there has been no follow-up study in other countries, so the relevance of these genes to NTG appears uncertain at present. Thus, we investigated the relationship between the HK2 and NCK2 genes and NTG in a Korean NTG cohort. METHODS: In total, 154 unrelated Korean patients with NTG and 101 normal Korean controls were recruited. Thus, a total of 255 participants were analyzed for NCK2 (rs2033008) and HK2 (rs678350) gene polymorphisms. RESULTS: The minor allele frequency (MAF) of rs678350 was significantly higher in NTG patients (MAF = 0.32) than in controls (MAF = 0.23) (OR, 1.586; 95% CI, 1.058 to 2.375; P = 0.028). This trend was more significant in the dominant model (OR, 1.908; 95% CI, 1.144 to 3.180; P = 0.015). When we performed logistic regression analysis to adjust for age, both the allelic and dominant models were still statistically significant. No significant difference was observed in rs2033008 allele or genotype frequencies between the NTG patients and control subjects. CONCLUSIONS: The current study suggested that HK2 gene polymorphism may contribute to the genetic susceptibility to NTG.

Effect of topical glaucoma medication on tear lipid layer thickness in patients with unilateral glaucoma.

Purpose: To compare the lipid layer thickness (LLT) using the LipiView® ocular surface interferometer (TearScience® Inc, Morrisville, NC) between the eye treated with glaucoma medication and untreated normal eye in the unilateral glaucoma patients, and evaluate the effect of topical glaucoma medication on the LLT parameters in glaucoma eyes. Methods: The participants in this cross-sectional comparative study were unilateral glaucoma patients treated with topical glaucoma medications for more than 12 months. Three LLT parameters (average, minimum, and maximum) obtained by the LipiView® were compared between the glaucomatous eye and normal eye. The factors associated with LLT parameters in the eyes treated with glaucoma medication were investigated with multiple regression analysis. Results: Thirty patients with unilateral normal tension glaucoma were enrolled in the present study. Lipid layer average, minimum, and maximum were 64.83 ± 16.50, 51.63 ± 16.73, and 82.53 ± 20.62 in glaucomatous eyes, 77.26 ± 17.81, 62.83 ± 20.99, and 86.13 ± 15.42 in normal eyes. Lipid layer average and minimum were significantly thinner than those in normal eyes (P < 0.001, P < 0.001, respectively). Longer duration of glaucoma eye drops and a greater number of glaucoma medications were associated with the lower LLT average (ß = -0.456, P < 0.001, ß = -8.517, P = 0.003, respectively), and increasing glaucoma medications have a significant correlation with lower LLT minimum in glaucoma eyes (ß = -8.814, P = 0.026). Conclusion: The present study highlights that patients with long-term glaucoma medications need to be assessed for LLT parameters objectively evaluate their ocular surface health.

Effect of Timolol on Optineurin Aggregation in Transformed Induced Pluripotent Stem Cells Derived From Patient With Familial Glaucoma.

Purpose: To determine a chemical agent that can reduce the aggregation of optineurin (OPTN) in cells differentiated from induced pluripotent stem cells obtained from a patient with normal-tension glaucoma (NTG) caused by an E50K mutation in the OPTN gene (OPTNE50K-NTG). Methods: Retinal ganglion cells (RGCs) were created from induced pluripotent stem cells derived from a healthy individual (wild-type [WT]-iPSCs) and from a patient with NTG due to OPTNE50K (E50K-iPSCs) mutation. The death of the induced RGCs was evaluated by counting the number of TUNEL- and ATH5-positive cells. Axonal growth was determined by measuring the axonal length of TUJ1-positive cells. OPTN aggregation was assessed by measuring the OPTN-positive area by immunofluorescence and by Western blotting. Autophagic flux assay was investigated by determining the light chain 3 (LC3)B-II/LC3B-I ratio and p62 expression by Western blotting. Results: The results showed OPTNE50K aggregation, activation of astrocytes, reduction in the number of RGCs, and enhancement of apoptotic cell death in the in vitro OPTNE50K model of NTG. Timolol was found to reduce the OPTNE50K-positive area and decreased the insoluble OPTNE50K, suggesting that it has the potential of reducing the OPTNE50K aggregation. Timolol also increased the ATH5-positive cells, decreased TUNEL-positive cells, increased the LC3B-II/LC3B-I ratio, and decreased the expression of p62. These findings suggest that timolol might enhance autophagic flux, leading to reduced OPTNE50K aggregation. Conclusions: Timolol should be considered a potential therapeutic agent specific to OPTNE50K-NTG because it can reduce the OPTNE50K aggregation in E50K-iPSCs-RGCs by enhancing autophagic flux and neuroprotective effects.

Topical Ripasudil Suppresses Retinal Ganglion Cell Death in a Mouse Model of Normal Tension Glaucoma.

Purpose: To assess if ripasudil has a neuroprotective effect using mice with excitatory amino acid carrier 1 (EAAC1) deletion (EAAC1 knockout [KO] mice), a mouse model of normal tension glaucoma. Methods: Topical administration (5 µL/day) of two different concentrations of ripasudil (0.4% and 2%) were applied to EAAC1 KO mice from 5 to 12 weeks old. Optical coherence tomography, multifocal electroretinograms, the measurement of intraocular pressure (IOP), and histopathology analyses were performed at 5, 8, and 12 weeks old. Retrograde labeling of retinal ganglion cells (RGCs), immunoblot, and immunohistochemical analyses of phosphorylated p38 mitogen-activated protein kinase (MAPK) in the retina were performed at 8 weeks old. Results: Topical ripasudil ameliorated retinal degeneration and improved visual function in EAAC1 KO mice at both 8 and 12 weeks old. Ripasudil reduced IOP and strongly suppressed the phosphorylation of p38 MAPK that stimulates RGC death in EAAC1 KO mice. Conclusions: These results suggest that, in addition to IOP reduction, ripasudil prevents glaucomatous retinal degeneration by neuroprotection, which is achieved by suppressing cell-death signaling pathways.

Autotaxin-Lysophosphatidic Acid Pathway in Intraocular Pressure Regulation and Glaucoma Subtypes.

Purpose: To compare the levels of autotaxin (ATX), lysophosphatidic acid (LPA), and lysophosphatidylcholine (LPC) in the aqueous humor (AH) of healthy control subjects with those of patients with different subtypes of glaucoma, and also to investigate the relationship of the ATX-LPA pathway with IOP and subtype of glaucoma. Methods: This study included 164 eyes of 164 consecutive cases of cataract and glaucoma surgery (37 healthy, 31 normal tension glaucoma, 49 primary open angle glaucoma, 28 secondary open angle glaucoma, and 19 exfoliation glaucoma). Aqueous levels of LPA, LPC, and ATX were quantified using liquid chromatography-tandem mass spectrometry and a two-site immunoenzymetric assay. The association between aqueous levels of ATX/LPA/LPC and IOP elevation in different glaucoma subtypes was investigated. The diagnostic values of indices of the ATX-LPA pathway were compared using receiver operating characteristic curve analysis. Results: Notable increases in ATX/LPA/LPC levels in glaucoma patients were observed. The ATX-LPA pathway was significantly related to IOP elevation and the subtype of glaucoma, especially in SOAG and XFG patients, and the area under the curve was significant for discriminating glaucoma eyes from healthy eyes. Conclusions: Bioactive ATX/LPA/LPC concentrations were present in aqueous humor, and higher ATX and LPA concentrations were significantly correlated with IOP in all study subjects. Furthermore, the ATX-LPA pathway was significantly related to glaucoma subtype. These results reveal the potentially important role of the ATX-LPA pathway for IOP regulation in healthy subjects and glaucoma patients.