Channelrhodopsins provide a breakthrough insight into strategies for curing blindness

Photoreceptor cells are the only retinal neurons that can absorb photons. Their degeneration due to some diseases or injuries leads to blindness. Retinal prostheses electrically stimulating surviving retinal cells and evoking a pseudo light sensation have been investigated over the past decade for restoring vision. Currently, a gene therapy approach is under development. Channelrhodopsin-2 derived from the green alga Chlamydomonas reinhardtii, is a microbial-type rhodopsin. Its specific characteristic is that it functions as a light-driven cation-selective channel. It has been reported that the channelrhodopsin-2 transforms inner light-insensitive retinal neurons to light-sensitive neurons. Herein, we introduce new strategies for restoring vision by using channelrhodopsins and discuss the properties of adeno-associated virus vectors widely used in gene therapy.     

rAAV-Mediated Subcellular Targeting of Optogenetic Tools in Retinal Ganglion Cells In Vivo

Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system.     

Looking within for vision

Channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii has been shown to be a directly light-switched cation-selective ion channel, which employs 11-cis retinal as its chromophore. This is the same chromophore as the mammalian photoreceptor's visual pigment-rhodopsin. Previously, investigators demonstrated that ChR2 can be used to optically control neuronal firing by depolarizing the cell. In this issue of Neuron, Bi et al. apply viral-mediated gene transfer to deliver ChR2 to retinal ganglion cells (RGC) in a rodent model of inherited blindness. In this way, the authors have genetically engineered surviving retinal neurons to take on the lost photoreceptive function. The conversion of light-insensitive retinal interneurons into photosensitive cells introduces an entirely new direction for treatments of blinding retinal degeneration.     

Visual Properties of Transgenic Rats Harboring the Channelrhodopsin-2 Gene Regulated by the Thy-1.2 Promoter

Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision.     

Calnexin is essential for rhodopsin maturation, Ca2+ regulation, and photoreceptor cell survival

In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration.     

Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation,Cell-Tailored Optogenetic Tool

Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell–specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.     

Loss of neuroprotective survival signal in mice lacking insulin receptor gene in rod photoreceptor cells

Insulin receptor (IR) signaling provides a trophic signal for transformed retinal neurons in culture, but the role of IR activity in vivo is unknown. We previously reported that light causes increased tyrosine phosphorylation of the IR in vivo, which leads to the downstream activation of the phosphoinositide 3-kinase and Akt pathway in rod photoreceptor cells. The functional role of IR in rod photoreceptor cells is not known. We observed that light stress induced tyrosine phosphorylation of the IR in rod photoreceptor cells, and we hypothesized that IR activation is neuroprotective. To determine whether IR has a neuroprotective role on rod photoreceptor cells, we used the Cre/lox system to specifically inactivate the IR gene in rod photoreceptors. Rod-specific IR knock-out mice have reduced the phosphoinositide 3-kinase and Akt survival signal in rod photoreceptors. The resultant mice exhibited no detectable phenotype when they were raised in dim cyclic light. However, reduced IR expression in rod photoreceptors significantly decreased retinal function and caused the loss of photoreceptors in mice exposed to bright light stress. These results indicate that reduced expression of IR in rod photoreceptor cells increases their susceptibility to light-induced photoreceptor degeneration. These data suggest that the IR pathway is important for photoreceptor survival and that activation of the IR may be an essential element of photoreceptor neuroprotection.     

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.     

Electrical Stimulation of Inner Retinal Neurons in Wild-Type and Retinally Degenerate (rd/rd) Mice

Electrical stimulation of the retina following photoreceptor degeneration in diseases such as retinitis pigmentosa and age-related macular degeneration has become a promising therapeutic strategy for the restoration of vision. Many retinal neurons remain functional following photoreceptor degeneration; however, the responses of the different classes of cells to electrical stimuli have not been fully investigated. Using whole-cell patch clamp electrophysiology in retinal slices we investigated the response to electrical stimulation of cells of the inner nuclear layer (INL), pre-synaptic to retinal ganglion cells, in wild-type and retinally degenerate (rd/rd) mice. The responses of these cells to electrical stimulation were extremely varied, with both extrinsic and intrinsic evoked responses observed. Further examination of the intrinsically evoked responses revealed direct activation of both voltage-gated Na⁺ channels and K⁺ channels. The expression of these channels, which is particularly varied between INL cells, and the stimulus intensity, appears to dictate the polarity of the eventual response. Retinally degenerate animals showed similar responses to electrical stimulation of the retina to those of the wild-type, but the relative representation of each response type differed. The most striking difference between genotypes was the existence of a large amplitude oscillation in the majority of INL cells in rd/rd mice (as previously reported) that impacted on the signal to noise ratio following electrical stimulation. This confounding oscillation may significantly reduce the efficacy of electrical stimulation of the degenerate retina, and a greater understanding of its origin will potentially enable it to be dampened or eliminated.     

Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia

Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemia-reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.     

The Wnt signaling pathway in retinal degenerations

The retina is a complex tissue composed of multiple interconnected cell layers, highly specialized for transforming light and color into electrical signals perceived by the brain. Damage or death of the primary light-sensing cells, the photoreceptors, results in devastating effects on vision. Despite the identification of numerous mutations that cause inherited retinal degenerations, the cellular and molecular mechanisms leading from the primary mutations to photoreceptor apoptosis are not understood. Wnt signaling has essential regulatory functions in a wide variety of critical developmental processes. Our research and others' have suggested that the Wnt pathway may be involved in retinal degeneration. Wnt ligands regulate developmental death of Drosophila photoreceptors, dysregulated Wnt signaling is involved in neuronal degeneration elsewhere in the central nervous system and Wnts control the expression of pro-survival growth factors in mammalian tissues. Additionally, altered expression of Wnt pathway genes, including the anti-apoptotic Wnt signaling regulator Dickkopf 3 (Dkk3), were observed during photoreceptor loss. This review examines the evidence and develops a model proposing a pro-survival role for Wnt signaling during photoreceptor injury. Because manipulating Wnt signaling has been demonstrated to have therapeutic potential for the treatment of Alzheimers disease, understanding the involvement of Wnts in photoreceptor death will determine whether targeting the Wnt pathway should also be considered as a possible therapeutic strategy for retinal degenerations.     

Decrease of opsin content in the developing rat photoreceptor cells by systemic administration of L-glutamate.

L-Glutamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. We purified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when 10 microliters or 15 microliters of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate-treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.     

Hydrogen sulfide protects the retina from light-induced degeneration by the modulation of Ca2+ influx

Hydrogen sulfide (H(2)S) has recently been recognized as a signaling molecule as well as a cytoprotectant. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are well-known as H(2)S-producing enzymes. We recently demonstrated that 3-mercaptopyruvate sulfurtransferase (3MST) along with cysteine aminotransferase (CAT) produces H(2)S in the brain and in vascular endothelium. However, the cellular distribution and regulation of these enzymes are not well understood. Here we show that 3MST and CAT are localized to retinal neurons and that the production of H(2)S is regulated by Ca(2+); H(2)S, in turn, regulates Ca(2+) influx into photoreceptor cells by activating vacuolar type H(+)-ATPase (V-ATPase). We also show that H(2)S protects retinal neurons from light-induced degeneration. The excessive levels of light exposure deteriorated photoreceptor cells and increased the number of TUNEL- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. Degeneration was greatly suppressed in the retina of mice administered with NaHS, a donor of H(2)S. The present study provides a new insight into the regulation of H(2)S production and the modulation of the retinal transmission by H(2)S. It also shows a cytoprotective effect of H(2)S on retinal neurons and provides a basis for the therapeutic target for retinal degeneration.     

Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

Photoreceptor degeneration after exposure of rats to incandescent illumination

Summary The retina of the albino rat undergoes degenerative changes when exposed to low intensity incandescent light. The retinal degeneration is limited specifically to the photoreceptor cells, and the pigment epithelium is unaffected. Early changes in the receptors included fragmentation of the inner and outer segments and pyknosis of the receptor cell nuclei. Phagocytic cells invaded and occupied the central retinal area of degeneration, between the receptor layer and the pigment epithelium, in the 4 and 5 day exposure periods. They were absent centrally after 14 and 30 days of exposure, but were present at these time periods in the peripheral retina, where photoreceptor destruction was still in progress. The destruction of photoreceptor cells, including the receptor and outer nuclear layers of the retina, by incandescent light progressed at a slightly reduced rate as compared to that after exposure to fluorescent light of the same intensity. These experiments indicate that exposure to either low intensity incandescent or fluorescent light will cause a selective degeneration of retinal photoreceptor cells, and therefore provide an easily reproducible model for the study of retinal structure and function in the absence of the receptors.This investigation was supported by Grants HD04102 and EY00595 from USPHS, and MH16077, a Research Scientist Award, to KVA, from the National Institute of Mental Health. Publication No. 1032, Department of Anatomy, Division of Basic Health Sciences, Emory University.The authors express their appreciation to Mrs. Sally Ware for her research assistance.