Takeshi Iwata

Glaucoma is an optic neuropathy characterized by progressive degeneration of retinal ganglion cells (RGCs) and visual loss. Although one of the highest risk factors for glaucoma is elevated intraocular pressure (IOP) and reduction in IOP is the only proven treatment, the mechanism of IOP regulation is poorly understood. We report that the P2Y ₆ receptor is critical for lowering IOP and that ablation of the P2Y ₆ gene in mice (P2Y ₆ KO) results in hypertensive glaucoma–like optic neuropathy. Topically applied uridine diphosphate, an endogenous selective agonist for the P2Y ₆ receptor, decreases IOP. The P2Y ₆ receptor was expressed in nonpigmented epithelial cells of the ciliary body and controlled aqueous humor dynamics. P2Y ₆ KO mice exhibited sustained elevation of IOP, age-dependent damage to the optic nerve, thinning of ganglion cell plus inner plexiform layers, and a reduction of RGC numbers. These changes in P2Y ₆ KO mice were attenuated by an IOP lowering agent. Consistent with RGC damage, visual functions were impaired in middle-aged P2Y ₆ KO mice. We also found that expression and function of P2Y ₆ receptors in WT mice were significantly reduced by aging, another important risk factor for glaucoma. In summary, our data show that dysfunctional purinergic signaling causes IOP dysregulation, resulting in glaucomatous optic neuropathy.

Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. The dry form is more common and accounts for about 85-90% of AMD patients in US, while Japanese AMD patients predominantly progress to wet-form or polypoidal choroidal vasculopathy (PCV). Recent studies have shown HTRA1, a serine protease gene, as major risk factor for wet form AMD (De Wan et al., Science 2006). Furthermore, we reported that the Japanese typical wet form AMD patients showed significant association with ARMS2/HTRA1 (Goto, Akahori et al., JOBDI 2009). The purpose of this study is to elucidate the function of ARMS2/HTRA1 gene promoter in wet-form AMD patients. The promoter sequence experiment showed that a great number of AMD patients had specific indel mutation in 3.8 kb upstream of HTRA1 gene. 2-3-fold increase of promoter activity was observed in indel HTRA1 promoter compared to control sequence (Iejima et al., JBC 2015). Furthermore, we created transgenic mice ubiquitously overexpressing mouse HtrA1 using the chicken act in promoter, continuous induction of HtrA1 in vivo was shown to lead to CNV, similar to wet AMD patients (Nakayama, Iejima et al., IOVS 2014). These results suggest that human HTRA1 expression is enhanced by AMD specific indel mutation in the promoter region of HTRA1 gene, and this enhanced HTRA1 may be concerned with induce retinal neovasucularization.

Purpose.: We determined the function of ARMS2 and HtrA1 in the choroid and retina using transgenic (Tg) mice and evaluated the effects of mainstream cigarette smoke on these mice. Methods.: The chicken actin promoter (CAG) was used to drive mouse HtrA1, human ARMS2, and ARMS2 (A69S) expression in the entire body of a mouse for one year. Fundus observations were performed with a Spectralis HRA+ optical coherence tomograph (OCT). Eyes were sectioned, stained with hematoxylin and eosin (H&E), and analyzed with immunohistochemistry. Mice were exposed to cigarette smoke for 30 min/d, 5 d/wk for 12 weeks using a mainstream smoking chamber (INH06-CIGR02A, MIPS). After 12 weeks, fundus observations and pathological analyses were performed. Results.: Approximately 18.2% of 12-month-old HtrA1 Tg mice exhibited choroidal neovascularization (CNV) by OCT and positive immunostaining with anti-CD31 and anti-fibronectin antibodies. Furthermore, elastic van Gieson (EVG) staining showed Bruch's membrane damage in HtrA1 Tg mice. No retinal changes were observed in ARMS2 and ARMS2 (A69S) Tg mice. A total of 12 weeks of exposure to mainstream cigarette smoke led to CNV rates of 7.7% for wild type (Wt) mice and 20% for HtrA1 Tg mice, but had no effect on ARMS2 Tg mice. In addition, abnormal deposits were observed between photoreceptor cells and the RPE in an HtrA1 Tg mouse exposed to mainstream cigarette smoke. Conclusions.: The HtrA1 overexpression and mainstream cigarette smoke can independently lead to CNV. The HtrA1 gene is a strong risk factor for wet AMD, but not all of the HtrA1 Tg mice developed CNV, suggesting that CNV development depends on multiple risk factors.

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population. In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed. Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation. This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.

More than 50 genes are reported as causative genes of autosomal recessive (ar) retinitis pigmentosa (RP) and cone-rod dystrophy (CRD). It is challenging to identify causative mutations for arRP and arCRD. The purpose of the present study was to investigate clinical and genetic features of two siblings with early-onset retinal dystrophy. Whole-exome sequencing was conducted for the two affected siblings and their unaffected brother and mother from a Japanese family. We performed complete ophthalmic examinations, including visual acuity, funduscopy, visual-field testing, electroretinography and optical coherence tomography. Whole-exome sequencing analysis identified novel compound heterozygous mutations, a splice site mutation (c.374 + 2T > C in intron 4) and a deletion mutation (c.575delC [p.T192MfsX28] in exon 6) of chromosome 8 open reading frame 37 (C8orf37) gene, which encodes a ciliary protein, in both patients. The mother carried the truncating mutation, and the brother carried neither mutation. Ophthalmic examinations revealed diffuse retinal degeneration, macular atrophy, non-recordable electroretinography responses, cataracts, and high myopia in both patients, who could not be diagnosed with either RP or CRD because of the severe retinal degeneration and early onset disease. Longitudinal follow-up of the patients revealed highly progressive retinal degeneration, macular atrophy, and visual field loss. Recessive C8orf37 mutations have been identified in early to adolescent-onset arRP and arCRD with macular involvement. Our study identified two novel truncating mutations of the C8orf37 gene in siblings with early-onset retinal dystrophy, macular atrophy, cataracts, and high myopia.

To compare a conventional cathode-ray tube (CRT) screen to organic light-emitting diode (OLED) and liquid crystal display (LCD) screens as visual stimulators to elicit multifocal electroretinograms (mfERGs), mfERGs were recorded from seven eyes of seven healthy volunteers (21 ± 2 years). The mfERGs elicited by a conventional CRT screen (S710, Compaq Computer Co.) were compared to those elicited by a studio-grade master OLED monitor (PVM-1741, Sony, Japan) and a conventional LCD (S1721, Flexscan, Eizo Nanao Corp., Japan). The luminance changes of each monitor were measured with a photodiode. CRT, OLED, and LCD screens with a frame frequency of 60 Hz were studied. A hexagonal stimulus array with 61 stimulus elements was created on each monitor. The serial white stimuli of the OLED screen at 60 Hz did not fuse, and that of the LCD screens fused. The amplitudes of P1 and P2 of the first-order kernels of the mfERGs were not significantly different from those elicited by the CRT and OLED screens, and the P1 amplitude of the first-order kernel elicited by the LCD stimuli was significantly smaller than that elicited by the CRT in all the groups of the averaged hexagonal elements. The implicit times were approximately 10 ms longer in almost all components elicited by the LCD screen compared to those elicited by the CRT screen. The mfERGs elicited by monitors other than the CRT should be carefully interpreted, especially those elicited by LCD screens. The OLED had good performance, and we conclude that it can replace the CRT as a stimulator for mfERGs; however, a collection of normative data is recommended.

To analyze the interaction of cortical potentials elicited by dichoptic stimulation of the dominant and fellow eyes at different frequencies, a pair of programmed power supply units were used to drive a light emitting diode (LED) mounted in the right and left eyes of light-proof goggles to elicit the visually evoked cortical responses (VECPs). The right eye was stimulated at 11.5 Hz and the left eye at 11.0 Hz. Then the stimulation was repeated with the frequency of stimulation switched to the other eyes. The stimulus duration was 5 ms. The sampling rate was 1.0 Hz, and the duration of collection was 200 ms. The VECP of each eye was extracted separately. Individual VECPs could be recorded separately after simultaneous dichoptic stimulation of each eye. The amplitudes of the VECPs were not significantly different after stimulating the dominant eye and the fellow eye separately. The implicit times of negative peak (N-2) and the second positive peak (P-2) were shorter after stimulation of the dominant eye than after stimulation of the fellow eye, but the difference was not significant. However, the implicit time of N-2 elicited by stimulating the dominant eye was significantly shorter when the stimulation rate was 11.5 Hz. The VECPs elicited by stimulating the two eyes can be recorded separately by simultaneous dichoptic stimulation. Dichoptic simultaneous stimulation required a shorter time and may be a more sensitive method of analyzing binocular interactions compared to the classic VECPs using monocular stimulation.

'Cone dystrophy with a supernormal rod electroretinogram (ERG)' is rare form of cone dystrophy, and no longitudinal description of the disease course has been reported in a Japanese population. Here, we describe long-term courses of 10 to 15 years in four Japanese patients with mutations in the KCNV2 gene. Four patients from three families were recruited. Two were siblings (Case 1, 24 y/o women; Case 2, 17 y/o man), and two were sporadic cases (Case 3, 17 y/o women; Case 4, 21 y/o women). All the patients presented with characteristic ERG findings. There were minimal abnormalities in fundus appearance: slight mottling of retinal pigment epithelium in the macula in all four cases, and granular change in the macula in Case 4. The visual acuity in Cases 1 and 2 did not change during the follow-up period, but the acuity in Cases 3 and 4 gradually decreased. Photoreceptor abnormalities in optical coherence tomography were found in all the cases, but were more severe in Cases 3 and 4. The long-term courses in Japanese patients were variable. The OCT was helpful in evaluating the disease progression.

Glaucoma is the leading cause for blindness affecting 60 million people worldwide. The optineurin (OPTN) E50K mutation was first identified in familial primary open-angle glaucoma (POAG), the onset of which is not associated with intraocular pressure (IOP) elevation, and is classified as normal-tension glaucoma (NTG). Optineurin (OPTN) is a multifunctional protein and its mutations are associated with neurodegenerative diseases such as POAG and amyotrophic lateral sclerosis (ALS). We have previously described an E50K mutation-carrying transgenic (E50K^-tg) mouse that exhibited glaucomatous phenotypes of decreased retinal ganglion cells (RGCs) and surrounding cell death at normal IOP. Further phenotypic analysis of these mice revealed persistent reactive gliosis and E50K mutant protein deposits in the outer plexiform layer (OPL). Over-expression of E50K in HEK293 cells indicated accumulation of insoluble OPTN in the endoplasmic reticulum (ER). This phenomenon was consistent with the results seen in neurons derived from induced pluripotent stem cells (iPSCs) from E50K mutation-carrying NTG patients. The E50K mutant strongly interacted with TANK-binding kinase 1 (TBK1), which prohibited the proper oligomerization and solubility of OPTN, both of which are important for OPTN intracellular transition. Treatment with a TBK1 inhibitor, BX795, abrogated the aberrant insolubility of the E50K mutant. Here, we delineated the intracellular dynamics of the endogenous E50K mutant protein for the first time and demonstrated how this mutation causes OPTN insolubility, in association with TBK1, to evoke POAG.