Akihiro Nishida

Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development

Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Here, using conditional gene ablation in mice, we show that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converted differentiating photoreceptor cells to amacrine-like neurons and led to a total lack of pinealocytes in the pineal gland. We also found that Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. Our results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue

We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

Functional Roles of Otx2 Transcription Factor in Postnatal Mouse Retinal Development

ABSTRACT We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/−; Crx−/− retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx−/− retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/−; Crx−/− retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.

The Significance of External Limiting Membrane Status for Visual Acuity in Age-Related Macular Degeneration

PURPOSE To evaluate status of the external limiting membrane (ELM) as a contributor of visual acuity (VA) in age-related macular degeneration (AMD). DESIGN Hospital-based, cross-sectional study. METHODS We retrospectively reviewed spectral-domain optical coherence tomography images of 158 patients with AMD who had undergone photodynamic therapy and classified them based on the status of the ELM: absent, discontinuous, or complete. We simultaneously assessed foveal thickness, presence or absence of subretinal fluid/mass, presence or absence of subretinal pigment epithelium fluid/mass, status of the inner segment/outer segment (IS/OS) junction, and status of the intermediate line between the IS/OS junction and retinal pigment epithelium. Correlation coefficients between each parameter and VA were analyzed. RESULTS There was a strong correlation between ELM status and VA (r = -0.75, P < .001), and that was higher than that of the IS/OS (r = -0.69, P < .001). Multivariate analysis showed that ELM status is the most important factor for VA. Other parameters that correlated with VA included age, status of the intermediate line, and presence of subretinal or subretinal pigment epithelium fibrosis. Foveal thickness showed V-shaped correlation, with the dividing line around 200 mum. CONCLUSION ELM status may be more useful than is IS/OS status in evaluation of retinal morphology and function in patients with AMD.

Function of Atypical Protein Kinase C λ in Differentiating Photoreceptors Is Required for Proper Lamination of Mouse Retina

The photoreceptor is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. To investigate the mechanisms of polarity formation of the photoreceptor cells, we conditionally knocked out atypical protein kinase Cλ (aPKCλ), which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptor cells using the Cre-loxP system. In aPKCλ conditional knock-out (CKO) mice, the photoreceptor cells displayed morphological defects and failed to form ribbon synapses. Intriguingly, lack of aPKCλ in differentiating photoreceptors led to severe laminar disorganization not only in the photoreceptor layer but also in the entire retina. Cell fate determination was not affected by total laminar disorganization. After Cre recombinase began to be expressed in the developing photoreceptors at embryonic day 12.5, both the immature photoreceptors and mitotic progenitors were dispersed throughout the CKO retina. We detected that adherens junction formation between the immature photoreceptors and the progenitors was lost in the CKO retina, whereas it was maintained between the progenitors themselves. These results indicate that the expression of aPKCλ in differentiating photoreceptors is required for total retinal lamination. Our data suggest that properly polarized photoreceptors anchor progenitors at the apical edge of the neural retina, which may be essential for building correct laminar organization of the retina.

Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment.

The purpose of this study was to evaluate the effects of the retinal environment and retinoic acid (RA) pretreatment on the differentiation of transplanted adult rat hippocampus-derived neural stem cells (AHSCs). AHSCs were transplanted into embryonic (E18) or neonatal (P6) rat retinal explants and the mixture was cultured for 2 weeks. Other AHSCs were stimulated by 0.5 microM all-trans RA for 6 days before transplantation. Immunofluorescent double staining showed that a larger number of AHSCs became beta-tubulin III-positive neurons in the E18 than in P6 retinas. In addition, many AHSCs became MAP2ab-positive and MAP5-positive neurons following RA pretreatment and transplantation. Only a few AHSCs became HPC-1-, calbindin-, PKC- or rhodopsin-positive cells under these conditions. We conclude that the microenvironment supplied by embryonic retinas is conductive to neuronal differentiation in general. RA stimulation before transplantation was also effective in stimulating differentiation.

Different characteristics of rat retinal progenitor cells from different culture periods

Embryonic retina is one of the possible cell sources that will repair degenerated retina such as retinitis pigmentosa. Retinal progenitor cells isolated from embryonic rats could be cultured and expanded in serum free medium with both epidermal growth factor and basic fibroblast growth factor. We analyzed the properties of two different retinal progenitor cells in terms of culture periods. Retinal progenitor cells from embryonic retina could be expanded keeping immature cell properties and had the ability to migrate into degenerated adult retina from subretinal space after transplantation. They differentiated into neurons and glias, even into photoreceptor cells both in vitro and in vivo. However, they appeared to lose their tissue specificity after a long-term culture.

Remission and dropout rate of anti-VEGF therapy for age-related macular degeneration.

Purpose. Anti-vascular endothelial growth factor (VEGF) therapy is a first-line treatment for age-related macular degeneration (AMD) but frequent visits and injections can be a burden for patients. The purpose of this study is to estimate the remission rate and tolerability of anti-VEGF therapy for AMD in a clinical setting. Methods. We investigated 90 eyes of 87 patients with AMD who underwent anti-VEGF therapy and were followed for more than 6 months. Ranibizumab and pegaptanib were used as anti-VEGF agents. Initial therapy was any of the following: a single injection, 3 consecutive monthly injections, or combination therapy with verteporfin. Additional injections were given as-needed during follow-up. An injection-free period greater than 6 months at the final observation was regarded as cessation; the reason for cessation was studied for each patient. Clinical characteristics were compared between patents with and without cessation. Results. The mean follow-up period was 12.8 months. Mean logMAR before and 6 months after the treatment was 0.89 and 0.83, respectively. Cessation was noted in 32 eyes of 31 patients (35.6%). Remission was achieved in 13 (40.6%) of these eyes. The other cases either did not wish to undergo further treatment or dropped out. Poor baseline visual acuity (VA) was associated with cessation. Conclusions. With current anti-VEGF therapy, remission was achieved in a limited number of AMD cases. The high frequency of voluntary cessation warrants consideration of an alternative treatment and/or supportive care for those with poor baseline VA.

Recurrence of polypoidal choroidal vasculopathy after photodynamic therapy

PurposeTo evaluate the recurrence of polypoidal lesions after photodynamic therapy (PDT) for polypoidal choroidal vasculopathy (PCV).MethodsThis is a retrospective review of data on 28 patients with PCV treated with PDT. The recurrent polypoidal lesions were observed with fluorescein and indocyanine green angiography.ResultsIn 26 of the 28 eyes the PCV remained inactive for more than 12 months after one to three treatments with PDT. Recurrence of the polypoidal lesions after more than a 12-month inactive period was reported in eight eyes: three at 15 months, two at 18 months, and three at 21 months. Kaplan-Meier estimates of recurrence were 11.5% at 15 months, 20.4% at 18 months, and 38.8% at 21 months, with no subsequent recurrence until passage of 36 months of inactivity after the last PDT. Additional PDT after recurrence improved or preserved visual acuity in 80% of those treated compared to the visual acuity before the first PDT.ConclusionsRecurrence of PCV occurs in about 40% of eyes treated for PCV even after a long period of inactivity, so careful follow-up is needed.

Determinants of Patient Satisfaction with Photodynamic Therapy for Neovascular Age-related Macular Degeneration or Polypoidal Choroidal Vasculopathy

PurposeTo evaluate the determinants of patient satisfaction with photodynamic therapy (PDT) for neovascular age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV).MethodsQuestionnaires were mailed to 69 patients who had undergone PDT for AMD or PCV at the Kobe City General Hospital. The questionnaire considered the following parameters: subjective change in visual acuity, subjective change in the relative scotoma in central vision, subjective change in visual distortion, and patient satisfaction with PDT scored on a 100-point scale.ResultsNine patients (14%) reported their subjective visual acuity change as “significantly improved” and 21 (32%) as “slightly improved”; 18 (27%) reported “no change”; 12 (18%) reported their visual acuity as “slightly worsened”; and 6 (9%) as “significantly worsened.” Subjective change in the central scotoma was reported improved in 43 patients (64%) and visual distortion had improved subjectively in 31 patients (47%). The satisfaction score was 59 ± 25 in patients who had undergone PDT for AMD and 75 ± 24 in those with PCV. Not only the visual acuity change but also the subjective change in central scotoma and visual distortion correlated significantly with the satisfaction score.ConclusionsMore patients who have undergone PDT for AMD or PCV perceive improvement in central scotoma and visual distortion than in visual acuity. Since these subjective changes correlated significantly with the satisfaction score, subjective change in central scotoma and visual distortion, in addition to visual acuity, should be taken into account in evaluating the benefits of PDT. Jpn J Ophthalmol 2007;51:368–374