Kiyoharu Miyagishima

Flow of energy in the outer retina in darkness and in light

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor’s synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

A Multiplex High-Throughput Gene Expression Assay to Simultaneously Detect Disease and Functional Markers in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

There is continuing interest in the development of lineage‐specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult‐like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage‐specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult‐like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high‐throughput format. Here, we use a multiplex high‐throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell‐derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell‐derived RPE that was then grown in 96‐ and 384‐well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell‐derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription‐polymerase chain reaction‐based measurements, confirming the ability of this high‐throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.

In Pursuit of Authenticity: Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for Clinical Applications

Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical‐basolateral polarity is fundamental for iPSC‐derived RPE (iPSC‐RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC‐RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC‐RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP‐dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady‐state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC‐RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC‐RPE derived for clinical applications.

Processing of S-cone signals in the inner plexiform layer of the mammalian retina.

Color information is encoded by two parallel pathways in the mammalian retina. One pathway compares signals from long- and middle-wavelength sensitive cones and generates red-green opponency. The other compares signals from short- and middle-/long-wavelength sensitive cones and generates blue-green (yellow) opponency. Whereas both pathways operate in trichromatic primates (including humans), the fundamental, phylogenetically ancient color mechanism shared among most mammals is blue-green opponency. In this review, we summarize the current understanding of how signals from short-wavelength sensitive cones are processed in the primate and nonprimate mammalian retina, with a focus on the inner plexiform layer where bipolar, amacrine, and ganglion cell processes interact to facilitate the generation of blue-green opponency.

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity

Background: A similar phototransduction cascade confers different light sensitivity in rods and cones. Results: Rod and cone Gαt are similar with respect to coupling to visual pigments and light-induced translocation. Conclusion: Rod and cone Gαt are equivalent functionally. Significance: Reduced sensitivity in cones does not result from reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones. The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ1γ1, 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.

Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods

The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of ∼200 seconds, but tip sensitivity recovered more slowly with a time constant of ∼680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is ∼1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.

Voltage‐sensitive conductances increase the sensitivity of rod photoresponses following pigment bleaching

Following substantial bleaching of the visual pigment, the desensitization of the rod photovoltage is not as substantial as the desensitization of the rod outer segment photocurrent. The block of cation conductances during the internal dialysis of Cs+ further desensitizes the photovoltage thereby eliminating its difference in desensitization with the rod outer segment photocurrent. Bleached visual pigment produced an acceleration of the rod photovoltage with respect to the outer segment photocurrent, which is eliminated upon internal dialysis of Cs+.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

SUMMARY Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications.