Light affects mitochondria to cause apoptosis to cultured cells: possible relevance to ganglion cell death in certain optic neuropathies

Retinal ganglion cell axons within the globe are laden with mitochondria that are unprotected from light (400–760 nm) impinging onto the retina. Light can be absorbed by mitochondrial enzymes such as cytochrome and flavin oxidases causing the generation of reactive oxygen species, and we have suggested this may pose a risk to ganglion cell survival if their energy state is compromised, as may be so in glaucoma or in Leber's Hereditary Optic Neuropathy. Here, we demonstrate that light (400–760 nm) provokes apoptosis in cultured retinal ganglion-5 cells, and that this effect is enhanced in low serum, and attenuated by various antioxidants. Apoptosis is shown to be caspase independent, involving reactive oxygen species generation and the activation of poly(ADP-ribose) polymerase-1 and apoptosis-inducing factor. We further show that light-induced apoptosis requires the participation of the mitochondrial respiratory chain. This was demonstrated by culturing fibroblasts (BJhTERT cells) in ethidium bromide for 40 days to deplete their mitochondrial DNA and perturb their mitochondrial respiratory chain function (BJhTERT rh0 cells). Only BJhTERT cells, with intact mitochondrial respiratory chain function were affected by light insult. Finally, we show that exposure of anaesthetized pigmented rat eye to white, but not red light, causes changes in the expression of certain retinal mRNAs (neurofilament light, Thy-1 and melanopsin) and optic nerve proteins (neurofilament light and tubulin), suggesting that ganglion cell survival is affected. Our findings support the proposal that the interaction of light, particularly the blue component, with intra-axonal ganglion cell mitochondria may be deleterious under certain circumstances, and suggest that reducing the light energy impinging upon the retina might benefit patients with certain optic neuropathies.     

Non-image-forming ocular photoreception in vertebrates

It has been accepted for a hundred years or more that rods and cones are the only photoreceptive cells in the retina. The light signals generated in rods and cones, after processing by downstream retinal neurons (bipolar, horizontal, amacrine and ganglion cells), are transmitted to the brain via the axons of the ganglion cells for further analysis. In the past few years, however, convincing evidence has rapidly emerged indicating that a small subset of retinal ganglion cells in mammals is also intrinsically photosensitive. Melanopsin is the signaling photopigment in these cells. The main function of the inner-retina photoreceptors is to generate and transmit non-image-forming visual information, although some role in conventional vision (image detection) is also possible.     

Melanopsin mediates retrograde visual signaling in the retina

The canonical flow of visual signals proceeds from outer to inner retina (photoreceptors→bipolar cells→ganglion cells). However, melanopsin-expressing ganglion cells are photosensitive and functional sustained light signaling to retinal dopaminergic interneurons persists in the absence of rods and cones. Here we show that the sustained-type light response of retinal dopamine neurons requires melanopsin and that the response is mediated by AMPA-type glutamate receptors, defining a retrograde retinal visual signaling pathway that fully reverses the usual flow of light signals in retinal circuits.     

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.     

Autocrine and paracrine interactions and neuroprotection in glaucoma

Retinal ganglion cells represent the output neurons of the retina. They are responsible for integrating electrical signals that originate with the photoreceptors and, via their axons that comprise the optic nerve, transmit that information to higher visual centers of the brain. The retinal ganglion cells reside on the inner surface of the retina and their axons course across the inner surface to exit at the back of the eye through a region known as the optic nerve head. Within this region, initiation of the degenerative processes associated with glaucoma are thought to occur, leading to degeneration of not only the optic nerve but also the retinal ganglion cells themselves. Studies aimed at understanding the mechanisms behind glaucoma have identified diverse cellular components and molecular events that occur in response to nerve injury. The challenge to date has been to identify and promote pro-survival events while suppressing those that support further degradation and loss of vision. Complicating this process is the fact that the cells and molecules involved can play multiple roles. An understanding of the players and their complex relationships is central to the development of a successful treatment strategy.     

Photochemical restoration of visual responses in blind mice

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are degenerative blinding diseases caused by the death of rods and cones, leaving the remainder of the visual system intact but largely unable to respond to light. Here, we show that AAQ, a synthetic small molecule photoswitch, can restore light sensitivity to the retina and behavioral responses in?vivo in mouse models of RP, without exogenous gene delivery. Brief application of AAQ bestows prolonged light sensitivity on multiple types of retinal neurons, resulting in synaptically amplified responses and center-surround antagonism in arrays of retinal ganglion cells (RGCs). Intraocular injection of AAQ restores the pupillary light reflex and locomotory light avoidance behavior in mice lacking retinal photoreceptors, indicating reconstitution of light signaling to brain circuits. AAQ and related photoswitch molecules present a potential drug strategy for restoring retinal function in degenerative blinding diseases.     

Retino-cortical information transmission achievable with a retina implant

Blind subjects with photoreceptor degeneration perceive phosphenes when their intact retinal ganglion cells are stimulated electrically. Is this approach suitable for transmitting enough information to the visual cortex for partially restoring vision? We stimulated the retina of anesthetized cats electrically and visually while recording the responses in the visual cortex. Transmission of retino-cortical information T was quantified by information theory. T was 20-160 bit/s (per stimulation and recording site) with random electrical or visual impulse stimulation at rates between 20 and 40 s-1. While increasing spatial density of independent electrical stimulation channels T did not saturate with 7 electrodes/mm2 retina. With seven electrodes up to 500 bit/s was transmitted to 15 cortical recording sites. Electrical stimulation basically employs temporal stimulus patterns. They are intimately linked with intensity/contrast information coded by the spike density of retinal ganglion cells. From the cortical information spread we estimated the spatial resolution as 0.5mm cortex corresponding to 0.5-1.0 degrees visual angle. If the human cortex can receive and decode the information transmitted by a retina implant, our quantitative results measured in cats suggest that visuo-motor coordination and object recognition in many in- and out-door situations will be possible.     

Distribution of Mammalian-Like Melanopsin in Cyclostome Retinas Exhibiting a Different Extent of Visual Functions

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.     

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.     

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.     

Pretreatment with Pyridoxamine Mitigates Isolevuglandin-associated Retinal Effects in Mice Exposed to Bright Light

The benefits of antioxidant therapy for treating age-related macular degeneration, a devastating retinal disease, are limited. Perhaps species other than reactive oxygen intermediates should be considered as therapeutic targets. These could be lipid peroxidation products, including isolevuglandins (isoLGs), prototypical and extraordinarily reactive γ-ketoaldehydes that avidly bind to proteins, phospholipids, and DNA and modulate the properties of these biomolecules. We found isoLG adducts in aged human retina but not in the retina of mice kept under dim lighting. Hence, to test whether scavenging of isoLGs could complement or supplant antioxidant therapy, we exposed mice to bright light and found that this insult leads to retinal isoLG-adduct formation. We then pretreated mice with pyridoxamine, a B₆ vitamer and efficient scavenger of γ-ketoaldehydes, and found that the levels of retinal isoLG adducts are decreased, and morphological changes in photoreceptor mitochondria are not as pronounced as in untreated animals. Our study demonstrates that preventing the damage to biomolecules by lipid peroxidation products, a novel concept in vision research, is a viable strategy to combat oxidative stress in the retina.     

Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina

To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.     

The autoregulation of retinal ganglion cell number

The development of the nervous system is dependent on a complex set of signals whose precise co-ordination ensures that the correct number of neurones are generated. This regulation is achieved through a variety of cues that influence both the generation and the maintenance of neurones during development. We show that in the chick embryo, stratified retinal ganglion cells (RGCs) are themselves responsible for providing the signals that control the number of RGCs that are generated, both by inhibiting the generation of new ganglion cells and by killing incoming migratory ganglion cells. Selective toxicological ablation of RGCs in the chick embryo resulted in the achronic generation of ganglion cells, which eventually led to the repopulation of the ganglion cell layer and a large decrease in the physiological cell death affecting postmitotic migratory neurones. Interestingly, the application of exogenous NGF reversed the effects of ganglion cell ablation on ganglion cell death. Because the only source of NGF in the retina is that produced by the stratified ganglion cells, we infer that these differentiated neurones regulate their own cell number by secreting NGF, a neurotrophin that has previously been shown to be responsible for the death of migrating ganglion cells.     

Rod vision is controlled by dopamine-dependent sensitization of rod bipolar cells by GABA

Dark and light adaptation of retinal neurons allow our vision to operate over an enormous light intensity range. Here we report a mechanism that controls the light sensitivity and operational range of rod-driven bipolar cells that mediate dim-light vision. Our data indicate that the light responses of these cells are enhanced by sustained chloride currents via GABA(C) receptor channels. This sensitizing GABAergic input is controlled by dopamine D1 receptors, with horizontal cells serving as a plausible source of GABA release. Our findings expand the role of dopamine in vision from its well-established function of suppressing rod-driven signals in bright light to enhancing the same signals under dim illumination. They further reveal a role for GABA in sensitizing the circuitry for dim-light vision, thereby complementing GABA's traditional role in providing dynamic feedforward and feedback inhibition in the retina.     

Requirement of p38 stress-activated MAP kinase for cell death in the developing retina depends on the stage of cell differentiation

The p38 members of the mitogen-activated protein kinase (MAPK) superfamily are activated by both environmental stress and endogenous signals, and may have either permissive or inhibitory roles upon both cell proliferation and cell death in the retina. We have previously shown that anisomycin, a protein synthesis inhibitor, and 2-aminopurine, a specific inhibitor of the double stranded-RNA dependent protein kinase, block apoptosis of ganglion cells induced by axotomy, and induce apoptosis of cells in the neuroblastic layer in developing rat retina. Using a specific inhibitor, we found that p38-stress activated MAP kinase is required for the death of post-mitotic cells induced by anisomycin, but not for the death of proliferating cells induced by 2-aminopurine, nor of axon-damaged retinal ganglion cells. We also show that p38 activation occurs either upstream of or parallel to the requirement for cyclic AMP to block apoptosis of post-mitotic cells, since the cyclic AMP-producing agent forskolin did not prevent p38 phosphorylation induced by anisomycin. Finally, the lack of immunostaining for phospho-p38 in apoptotic profiles suggests that p38 activation does not kill retinal cells directly, but more likely through the mediation of neighboring cells.     

The Notch signaling pathway in retinal dysplasia and retina vascular homeostasis

The retina is one of the most essential elements of vision pathway in vertebrate. The dysplasia of retina cause congenital blindness or vision disability in individuals, and the misbalance in adult retinal vascular homeostasis leads to neovaseularization-associated diseases in adults, such as diabetic retinopathy or age-related macular degeneration. Many developmental signaling pathways are involved in the process of retinal development and vascular homeostasis. Among them, Notch signaling pathway has long been studied, and Notch signaling-interfered mouse models show both neural retina dysplasia and vascular abnormality. In this review, we discuss the roles of Notch signaling in the maintenance of retinal progenitor cells, specification of retinal neurons and glial cells, and the sustaining of retina vascular homeostasis, especially from the aspects of conditional knockout mouse models. The potential of Notch signal mampulation may provide a powerful cell fate- and neovascularization-controlling tool that could have important applications in la'eatment of retinal diseases.     

Light-dependent Structural Change of Chicken Retinal Cryptochrome4

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells. We further developed several monoclonal antibodies to the carboxyl-terminal extension of cCRY4 and localized cCRY4 protein with immunohistochemistry. Consistent with the results of in situ hybridization, cCRY4 immunoreactivity was found in visual pigment cells and cells located at the inner nuclear layer and the retinal ganglion cell layer. Among the antibodies, one termed C1-mAb had its epitope within the carboxyl-terminal 14-amino acid sequence (QLTRDDADDPMEMK) and associated with cCRY4 in the retinal soluble fraction more strongly in the dark than under blue light conditions. Immunoprecipitation experiments under various light conditions indicated that cCRY4 from the immunocomplex formed in the dark dissociated from C1-mAb during blue light illumination as weak as 25 μW/cm² and that the release occurred with not only blue but also near UV light. These results suggest that cCRY4 reversibly changes its structure within the carboxyl-terminal region in a light-dependent manner and operates as a photoreceptor or magnetoreceptor with short wavelength sensitivity in the retina.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Extracellular recording in neuronal networks with substrate integrated microelectrode arrays

A photolithographically produced array of 60 substrate-integrated microelectrodes was used for extracellular recording. Neuronal electrical activity was recorded from chicken retinal ganglion cells with or without stimulation by diffuse light. The retina was removed from chicken embryos of embryonic day 14–18. Only cells recorded from day 18 retina would react to photostimulation, increasing their activity when stimulated, corresponding to the developmental time course of photoreceptor differentiation.     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.