Detailed Evaluation of Chromatic Pupillometry and Full-Field Stimulus Testing to Assess Ultralow Vision in Retinitis Pigmentosa.

Purpose: Novel therapeutic options, such as regenerative medicine and gene therapy, are now emerging as viable treatment options for patients with severe visual impairments, such as retinitis pigmentosa (RP). Gradable assessment of patients' visual function is essential to consider treatment options and to evaluate treatment outcomes; however, evaluation of visual function in patients with advanced low vision is often challenging because of patients' poor and sometimes unpredictable responses. In this study, we attempted to accurately assess visual capabilities and disease stage in patients with RP with a visual acuity (VA) of ≤ 0.01. Design: Retrospective analysis of visual function indicators, including VA, retinal thickness, full-field stimulus testing (FST), and chromatic pupillometry. Subjects: Overall, 43 patients (84 eyes) with advanced RP with a VA of ≤ 0.01 visited Kobe City Eye Hospital from 2019 to 2021. Methods: Hierarchical (multilevel) Bayesian modeling was used to estimate individual eye's pupil response and FST threshold, taking into account the ambiguity and randomness often observed in patients with ultralow vision. Using the estimated ability obtained from each test, the correlation between each test and retinal thickness was further analyzed to make a comprehensive assessment of the data. Main Outcome Measures: Visual acuity, retinal thickness, FST threshold, and pupil diameter change to different light stimuli. Results: Full-field stimulus testing and pupillometry measurements were moderately correlated with VA but exhibited a wide range of values within the same VA groups. Full-field stimulus testing was not correlated with central retinal thickness at counting fingers/hand motion VA range and seemed to reflect overall remaining photoreceptor function, including peripheral retina. Pupillometry may be able to distinguish between different levels of inner retinal function. Conclusions: The combination of pupillometry and FST allowed for graded evaluation of visual function within patients grouped in the same VA groups in patients with advanced RP with ultralow vision. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Competency of iPSC-derived retinas in MHC-mismatched transplantation in non-human primates.

Transplantation of embryonic/induced pluripotent stem cell-derived retina (ESC/iPSC-retina) restores host retinal ganglion cell light responses in end-stage retinal degeneration models with host-graft synapse formation. We studied the immunological features of iPSC-retina transplantation using major histocompatibility complex (MHC)-homozygote monkey iPSC-retinas in monkeys with laser-induced retinal degeneration in MHC-matched and -mismatched transplantation. MHC-mismatched transplantation without immune suppression showed no evident clinical signs of rejection and histologically showed graft maturation without lymphocytic infiltration, although immunological tests using peripheral blood monocytes suggested subclinical rejection in three of four MHC-mismatched monkeys. Although extensive photoreceptor rosette formation was observed on histology, evaluation of functional integration using mouse models such as mouse ESC-retina (C57BL/6) transplanted into rd1(C3H/HeJ, MHC-mismatched model) elicited light responses in the host retinal ganglion cells after transplantation but with less responsiveness than that in rd1-2J mice (C57BL/6, MHC-matched model). These results suggest the reasonable use of ESC/iPSC-retina in MHC-mismatched transplantation, albeit with caution.

A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation.

Pluripotent stem cell (PSC)-derived retinal sheet transplanted in vivo can form structured photoreceptor layers, contact with host bipolar cells, and transmit light signals to host retinas. However, a major concern is the presence of graft bipolar cells that may impede host-graft interaction. In this study, we used human ESC-retinas with the deletion of Islet-1 (ISL1) gene to achieve the reduced graft ON-bipolar cells after xenotransplantation into end-stage retinal degeneration model rats. Compared with wild-type graft, ISL1 -/- hESC-retinas showed better host-graft contact, with indication of host-graft synapse formation and significant restoration of light responsiveness in host ganglion cells. We further analyzed to find out that improved functional integration of ISL1 -/- hESC-retinas seemed attributed by a better host-graft contact and a better preservation of host inner retina. ISL1 -/- hESC-retinas are promising for the efficient reconstruction of a degenerated retinal network in future clinical application.

Polymorphism in the symmetries of gastric pouch arrangements in the sea anemone D. lineata.

Symmetry in the arrangement of body parts is a distinctive phylogenetic feature of animals. Cnidarians show both bilateral and radial symmetries in their internal organs, such as gastric pouches and muscles. However, how different symmetries appear during the developmental process remains unknown. Here, we report intraspecific variations in the symmetric arrangement of gastric pouches, muscles, and siphonoglyphs, the Anthozoan-specific organ that drives water into the organism, in D. lineata (Diadumenidae, Actiniaria). We found that the positional arrangement of the internal organs was apparently constrained to either biradial or bilateral symmetries depending on the number of siphonoglyphs. Based on the morphological observations, a mathematical model of internal organ positioning was employed to predict the developmental backgrounds responsible for the biradial and bilateral symmetries. In the model, we assumed that the specification of gastric pouches is orchestrated by lateral inhibition and activation, which results in different symmetries depending on the number of siphonoglyphs. Thus, we propose that a common developmental program can generate either bilateral or biradial symmetries depending on the number of siphonoglyphs formed in the early developmental stages.

Genetically engineered stem cell-derived retinal grafts for improved retinal reconstruction after transplantation.

ESC/iPSC-retinal sheet transplantation, which supplies photoreceptors as well as other retinal cells, has been shown to be able to restore visual function in mice with end-stage retinal degeneration. Here, by introducing a novel type of genetically engineered mouse ESC/iPSC-retinal sheet with reduced numbers of secondary retinal neurons but intact photoreceptor cell layer structure, we reinforced the evidence that ESC/iPSC-retinal sheet transplantation can establish synaptic connections with the host, restore light responsiveness, and reduce aberrant retinal ganglion cell spiking in mice. Furthermore, we show that genetically engineered grafts can substantially improve the outcome of the treatment by improving neural integration. We speculate that this leads to reduced spontaneous activity in the host which in turn contributes to a better visual recovery.

[Toward establishment of regenerative cell therapy for retinitis pigmentosa using iPS cell derived retinal sheet].

Retinitis pigmentosa (RP) is a group of hereditary diseases that involve loss of photoreceptors. There has been no established treatment for RP, and it is now the 2nd leading cause of blindness in Japan. Previous clinical researches using human fetal retina transplantation suggested some functional recovery in vision, but it did not become a standard therapy because of ethical concerns for using fetus tissues. Invention of induced pluripotent stem cells (iPSC) in 2006 and the establishment of retinal organoids induction protocol from ES/iPS cells have paved a way of cell therapy for RP without ethical concerns. Our team has shown that mouse iPSC derived retinas can survive and mature after subretinal transplantation to the end-stage retinal degeneration model mice. Further, human ESC derived retinas survived and matured in retinal degeneration monkey models. Recently, we have established a qualitative and quantitative evaluation tool for photoreceptor synapses, QUANTOS, and showed that photoreceptors in mouse iPSC derived retina can form photoreceptor synapses in a time dependent manner after transplantation. We are now moving toward 1st in human clinical trial using iPSC derived retina for RP.

Multielectrode Array Recording of Mouse Retinas Transplanted with Stem Cell-Derived Retinal Sheets.

Retinal multielectrode array (MEA) recording allows us to examine the action potentials of retinal ganglion cells and field potentials of photoreceptors and bipolar cells. In addition to studying the retinal circuitry, it has become one of the standard examination tools for the characterization of stem cell-derived retinal transplantation in degenerated retinas. Besides the detection of responses to simple light stimulation, it is also necessary to consider the spatial correlation of the graft and the electrodes, in order to unbiasedly reveal the locally reconstructed retinal circuitry after transplantation. Here, we introduce our newly developed protocol of MEA recording and analysis that may serve as a standard for evaluating transplanted retinas.

Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders.

Medium- to long-term survival and functional examination of human iPSC-derived retinas in rat and primate models of retinal degeneration.

BACKGROUND: We have previously reported that xeno-transplanted human ESC-derived retinas are able to mature in the immunodeficient retinal degeneration rodent models, similar to allo-transplantations using mouse iPSC-derived retina. The photoreceptors in the latter developed outer segments and formed synapses with host bipolar cells, driving light responses of host retinal ganglion cells. In view of clinical application, here we further confirmed the competency of human iPSC-derived retina (hiPSC-retina) to mature in the degenerated retinas of rat and monkey models. METHODS: Human iPSC-retinas were transplanted in rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats and two monkeys with laser-induced photoreceptor degeneration. Graft maturation was studied by immunohistochemistry and its function was examined by multi-electrode array (MEA) recording in rat retinas and visually-guided saccade (VGS) in a monkey. FINDINGS: A substantial amount of mature photoreceptors in hiPSC-retina graft survived well in the host retinas for at least 5 months (rat) to over 2 years (monkey). In 4 of 7 transplanted rat retinas, RGC light responses were detected at the grafted area. A mild recovery of light perception was also suggested by the VGS performance 1.5 years after transplantation in that monkey. INTERPRETATION: Our results support the competency of hiPSC-derived retinas to be clinically applied for transplantation therapy in retinal degeneration, although the light responses observed in the present models were not conclusively distinguishable from residual functions of degenerating host retinas. The functional analysis may be further elaborated using other models with more advanced retinal degeneration.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation.

Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets.

Alternative Formation of Red-Shifted Channelrhodopsins: Noncovalent Incorporation with Retinal-Based Enamine-Type Schiff Bases and Mutated Channelopsin.

Red-shifted channelrhodopsins (ChRs) are attractive for optogenetic tools. We developed a new type of red-shifted ChRs that utilized noncovalent incorporation of retinal and 3,4-dehydroretinal-based enamine-type Schiff bases and mutated channelopsin, C1C2-K296G. These ChRs exhibited absorption maxima that were shifted 10-30 nm toward longer wavelengths than that of C1C2-ChR regenerated with all-trans-retinal.

Origin of the low thermal isomerization rate of rhodopsin chromophore.

Low dark noise is a prerequisite for rod cells, which mediate our dim-light vision. The low dark noise is achieved by the extremely stable character of the rod visual pigment, rhodopsin, which evolved from less stable cone visual pigments. We have developed a biochemical method to quickly evaluate the thermal activation rate of visual pigments. Using an isomerization locked chromophore, we confirmed that thermal isomerization of the chromophore is the sole cause of thermal activation. Interestingly, we revealed an unexpected correlation between the thermal stability of the dark state and that of the active intermediate MetaII. Furthermore, we assessed key residues in rhodopsin and cone visual pigments by mutation analysis and identified two critical residues (E122 and I189) in the retinal binding pocket which account for the extremely low thermal activation rate of rhodopsin.

Photochemical properties of mammalian melanopsin.

Melanopsin is the photoreceptor molecule of intrinsically photosensitive retinal ganglion cells, which serve as the input for various nonvisual behavior and physiological functions fundamental to organisms. The retina, therefore, possess a melanopsin-based nonvisual system in addition to the visual system based on the classical visual photoreceptor molecules. To elucidate the molecular properties of melanopsin, we have exogenously expressed mouse melanopsin in cultured cells. We were able to obtain large amounts of purified mouse melanopsin and conducted a comprehensive spectroscopic study of its photochemical properties. Melanopsin has an absorption maximum at 467 nm, and it converts to a meta intermediate having an absorption maximum at 476 nm. The melanopsin photoreaction is similar to that of squid rhodopsin, exhibiting bistability that results in a photosteady mixture of a resting state (melanopsin containing 11-cis-retinal) and an excited state (metamelanopsin containing all-trans-retinal) upon sustained irradiation. The absorption coefficient of melanopsin is 33000 ± 1000 M(-1) cm(-1), and its quantum yield of isomerization is 0.52; these values are also typical of invertebrate bistable pigments. Thus, the nonvisual system in the retina relies on a type of photoreceptor molecule different from that of the visual system. Additionally, we found a new state of melanopsin, containing 7-cis-retinal (extramelanopsin), which forms readily upon long-wavelength irradiation (yellow to red light) and photoconverts to metamelanopsin with short-wavelength (blue light) irradiation. Although it is unclear whether extramelanopsin would have any physiological role, it could potentially allow wavelength-dependent regulation of melanopsin functions.

The C-terminus of the G protein α subunit controls the affinity of nucleotides.

The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.

Covalent bond between ligand and receptor required for efficient activation in rhodopsin.

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.

Evolution of opsins and phototransduction.

Opsins are the universal photoreceptor molecules of all visual systems in the animal kingdom. They can change their conformation from a resting state to a signalling state upon light absorption, which activates the G protein, thereby resulting in a signalling cascade that produces physiological responses. This process of capturing a photon and transforming it into a physiological response is known as phototransduction. Recent cloning techniques have revealed the rich and diverse nature of these molecules, found in organisms ranging from jellyfish to humans, functioning in visual and non-visual phototransduction systems and photoisomerases. Here we describe the diversity of these proteins and their role in phototransduction. Then we explore the molecular properties of opsins, by analysing site-directed mutants, strategically designed by phylogenetic comparison. This site-directed mutant approach led us to identify many key features in the evolution of the photoreceptor molecules. In particular, we will discuss the evolution of the counterion, the reduction of agonist binding to the receptor, and the molecular properties that characterize rod opsins apart from cone opsins. We will show how the advances in molecular biology and biophysics have given us insights into how evolution works at the molecular level.