Specific Retinal Cell Types Research Articles

Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

Restricted oxygen supply in the aging eye may lead to hypoxic conditions in the outer retina and contribute not only to physiological aging but also to nonhereditary degenerative retinal diseases. To understand the hypoxic response of specific retinal cell types, we performed single-cell RNA sequencing of retinas isolated from mice exposed to hypoxia. Significantly upregulated expression of marker genes in hypoxic clusters confirmed a general transcriptional response to hypoxia. By focusing on the hypoxic response in photoreceptors, we identified and confirmed a kinesin motor protein (Kif4) that was specifically and strongly induced in hypoxic cones. In contrast, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters but demonstrated isoform switching in hypoxia. The resulting short variants of these gene transcripts are connected to epitranscriptomic regulation, a notion supported by the differential expression of writers, readers and erasers of m6A RNA methylations in the hypoxic retina. Our data indicate that retinal cells adapt to hypoxic conditions by adjusting their transcriptome at various levels including gene expression, alternative splicing and the epitranscriptome. Adaptational processes may be cell-type specific as exemplified by the cone-specific upregulation of Kif4 or general like alternative splicing of RNA binding proteins.

Artificial Visual Information Produced by Retinal Prostheses.

Numerous retinal prosthetic systems have demonstrated somewhat useful vision can be restored to individuals who had lost their sight due to outer retinal degenerative diseases. Earlier prosthetic studies have mostly focused on the confinement of electrical stimulation for improved spatial resolution and/or the biased stimulation of specific retinal ganglion cell (RGC) types for selective activation of retinal ON/OFF pathway for enhanced visual percepts. To better replicate normal vision, it would be also crucial to consider information transmission by spiking activities arising in the RGC population since an incredible amount of visual information is transferred from the eye to the brain. In previous studies, however, it has not been well explored how much artificial visual information is created in response to electrical stimuli delivered by microelectrodes. In the present work, we discuss the importance of the neural information for high-quality artificial vision. First, we summarize the previous literatures which have computed information transmission rates from spiking activities of RGCs in response to visual stimuli. Second, we exemplify a couple of studies which computed the neural information from electrically evoked responses. Third, we briefly introduce how information rates can be computed in the representative two ways – direct method and reconstruction method. Fourth, we introduce in silico approaches modeling artificial retinal neural networks to explore the relationship between amount of information and the spiking patterns. Lastly, we conclude our review with clinical implications to emphasize the necessity of considering visual information transmission for further improvement of retinal prosthetics.

Current perspectives in Leber congenital amaurosis type 8 mouse modeling.

Mutations in the CRB1 (Crumbs homolog 1) cause rare retinal diseases like retinitis pigmentosa type 12 (RP12) and Leber congenital amaurosis type 8 (LCA8). RP12 results in progressively worsening peripheral vision, whereas LCA8 causes severe visual impairment at birth or in early life. While several mouse models have been proposed for RP12, few replicate the full spectrum of human LCA8 pathology, such as disorganized retinal layering, abnormal retinal thickening, pigmentary defects, hyperreflective lesions, and severely attenuated electroretinogram responses at birth. Six models have been proposed utilizing the Cre-loxP system to delete candidate genes in specific retinal cell types and developmental stages. The model ablating Crb1 and its homolog Crb2 (using mRx-Cre) from the beginning of the eye development is the most complete as it shows blindness during the eye-opening stage, pigmentary defects in the RPE, ganglion cell layer heterotopia, disruption of retinal lamination, and acellular patches. LCA8 represents a unique type of retinal dystrophy among LCA subtypes, driven by dysfunctional retinal progenitor cells during eye development. In contrast, other LCA types and RP12 are caused by photoreceptor defects. Therefore, the most accurate LCA8-like mouse model must target both alleles of the Crb1 and Crb2 genes in the optic vesicle or earlier.

Role of mTORC1 activity during early retinal development and lamination in human-induced pluripotent stem cell\u2010derived retinal organoids

Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.

Embryonic hyperglycemia perturbs the development of specific retinal cell types, including photoreceptors.

Diabetes is linked to various long-term complications in adults, such as neuropathy, nephropathy and diabetic retinopathy. Diabetes poses additional risks for pregnant women, because glucose passes across the placenta, and excess maternal glucose can result in diabetic embryopathy. While many studies have examined the teratogenic effects of maternal diabetes on fetal heart development, little is known about the consequences of maternal hyperglycemia on the development of the embryonic retina. To address this question, we investigated retinal development in two models of embryonic hyperglycemia in zebrafish. Strikingly, we found that hyperglycemic larvae displayed a significant reduction in photoreceptors and horizontal cells, whereas other retinal neurons were not affected. We also observed reactive gliosis and abnormal optokinetic responses in hyperglycemic larvae. Further analysis revealed delayed retinal cell differentiation in hyperglycemic embryos that coincided with increased reactive oxygen species (ROS). Our results suggest that embryonic hyperglycemia causes abnormal retinal development via altered timing of cell differentiation and ROS production, which is accompanied by visual defects. Further studies using zebrafish models of hyperglycemia will allow us to understand the molecular mechanisms underlying these effects.

Correlation between small-scale methylation changes and gene expression during the development of myopia.

Visually induced changes in the expression of early growth response-1 (EGR1), FBJ osteosarcoma oncogene (FOS), and NGFI-A binding protein-2 (NAB2) appear to form a part of a retinal network fundamental to ocular growth regulation, and thus, the development of myopia (short-sightedness). However, it is unclear how environmental (visual) cues are translated into these molecular changes. One possibility is through epigenetic modifications such as DNA methylation, a known regulator of such processes. By sequencing bisulfite-converted DNA amplicons, this study examined whether changes in DNA methylation occur within specific regulatory and promoter regions of EGR1, FOS, and NAB2 during the periods of increased and decreased ocular growth in chicks. Visually induced changes in ocular growth rates were associated with single-point, but not large-scale, shifts in methylation levels within the investigated regions. Analysis of methylation pattern variability (entropy) demonstrated that the observed methylation changes are occurring within small subpopulations of retinal cells. This concurs with previous observations that EGR1 and FOS are differentially regulated at the peptide level within specific retinal cell types. Together, the findings of this study support a potential role for DNA methylation in the translation of external visual cues into molecular changes critical for ocular growth regulation and myopia development.

Evidence of regional specializations in regenerated zebrafish retina

Adult zebrafish are capable of functional retinal regeneration following damage. A goal of vision science is to stimulate or permit a similar process in mammals to treat human retinal disease and trauma. Ideally such a process would reconstitute the stereotyped, two-dimensional topographic patterns and regional specializations of specific cell types, functionally important for representation of the visual field. An example in humans is the cone-rich fovea, essential for high-acuity color vision. Stereotyped, global topographic patterns of specific retinal cell types are also found in zebrafish, particularly for cone types expressing the tandemly-replicated lws (long wavelength-sensitive) and rh2 (middle wavelength-sensitive) opsins. Here we examine whether regionally specialized patterns of LWS1 and LWS2 cones are restored in regenerated retinas in zebrafish. Adult transgenic zebrafish carrying fluorescent reporters for lws1 and lws2 were subjected to retinal lesions that destroy all neurons but spare glia, via intraocular injection of the neurotoxin ouabain. Regenerated and contralateral control retinas were mounted whole or sectioned, and imaged. Overall spatial patterns of lws1 vs. lws2 opsin-expressing cones in regenerated retinas were remarkably similar to those of control retinas, with LWS1 cones in ventral/peripheral regions, and LWS2 cones in dorsal/central regions. However, LWS2 cones occupied a smaller fraction of regenerated retina, and several cones co-expressed the lws1 and lws2 reporters in regenerated retinas. Local patterns of regenerated LWS1 cones showed modest reductions in regularity. These results suggest that some of the regional patterning information, or the source of such signals, for LWS cone subtypes may be retained by undamaged cell types (Müller glia or RPE) and re-deployed during regeneration.

Chimeric Helper-Dependent Adenoviruses Transduce Retinal Ganglion Cells and Müller Cells in Human Retinal Explants.

Purpose: Despite numerous recent advances in retinal gene therapy using adeno-associated viruses (AAVs) as delivery vectors, there remains a crucial need to identify viral vectors with the ability to transduce specific retinal cell types and that have a larger carrying capacity than AAV. In this study, we evaluate the retinal tropism of 2 chimeric helper-dependent adenoviruses (HDAds), helper-dependent adenovirus serotype 5 (HDAd5)/3 and HDAd5/35, both ex vivo using human retinal explants and in vivo using rats. Methods: We transduced cultured human retinal explants with HDAd5/3 and HDAd5/35 carrying an eGFP vector and evaluated tropism and transduction efficiency using immunohistochemistry. To assess in vivo transduction efficiency, subretinal injections were performed in wild-type Sprague-Dawley rats. For both explants and subretinal injections, we delivered 10 μL (1 × 106 vector genomes/mL) and assessed tropism at 7- and 14-days post-transduction, respectively. Results: HDAd5/3 and HDAd5/35 both transduced human retinal ganglion cells (RGCs) and Müller cells, but not photoreceptors, in human retinal explants. However, subretinal injections in albino rats resulted in transduction of the retinal pigmented epithelium only, highlighting species-specific differences in retinal tropism and the value of a human explant model when testing vectors for eventual human gene therapy. Conclusions: Chimeric HDAds are promising candidates for the delivery of large genes, multiple genes, or neuroprotective factors to Müller cells and RGCs. These vectors may have utility for targeted therapy of neurodegenerative diseases primarily involving retinal ganglion or Müller cell types, such as glaucoma or macular telangiectasia type 2.

Retinal ganglion cell dysfunction in mice following acute intraocular pressure is exacerbated by P2X7 receptor knockout

There is increasing evidence for the vulnerability of specific retinal ganglion cell (RGC) types in those with glaucoma and in animal models. In addition, the P2X7-receptor (P2X7-R) has been suggested to contribute to RGC death following stimulation and elevated IOP, though its role in RGC dysfunction prior to death has not been examined. Therefore, we examined the effect of an acute, non-ischemic intraocular pressure (IOP) insult (50 mmHg for 30 min) on RGC function in wildtype mice and P2X7-R knockout (P2X7-KO) mice. We examined retinal function using electroretinogram recordings and individual RGC responses using multielectrode arrays, 3 days following acute IOP elevation. Immunohistochemistry was used to examine RGC cell death and P2X7-R expression in several RGC types. Acute intraocular pressure elevation produced pronounced dysfunction in RGCs; whilst other retinal neuronal responses showed lesser changes. Dysfunction at 3 days post-injury was not associated with RGC loss or changes in receptive field size. However, in wildtype animals, OFF-RGCs showed reduced spontaneous and light-elicited activity. In the P2X7-KO, both ON- and OFF-RGC light-elicited responses were reduced. Expression of P2X7-R in wildtype ON-RGC dendrites was higher than in other RGC types. In conclusion, OFF-RGCs were vulnerable to acute IOP elevation and their dysfunction was not rescued by genetic ablation of P2X7-R. Indeed, knockout of P2X7-R also caused ON-RGC dysfunction. These findings aid our understanding of how pressure affects RGC function and suggest treatments targeting the P2X7-R need to be carefully considered.

New Technologies to Study Functional Genomics of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in people over 50 years old in developed countries. Currently, we still lack a comprehensive understanding of the genetic factors contributing to AMD, which is critical to identify effective therapeutic targets to improve treatment outcomes for AMD patients. Here we discuss the latest technologies that can facilitate the identification and functional study of putative genes in AMD pathology. We review improved genomic methods to identify novel AMD genes, advances in single cell transcriptomics to profile gene expression in specific retinal cell types, and summarize recent development of in vitro models for studying AMD using induced pluripotent stem cells, organoids and biomaterials, as well as new molecular technologies using CRISPR/Cas that could facilitate functional studies of AMD-associated genes.

Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types.

PurposeEarly in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist.MethodsCRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene.ResultsRetinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types.Conclusions and Translational RelevanceThe PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.

Retinal miRNA Functions in Health and Disease.

The health and function of our visual system relies on accurate gene expression. While many genetic mutations are associated with visual impairment and blindness, we are just beginning to understand the complex interplay between gene regulation and retinal pathologies. MicroRNAs (miRNAs), a class of non-coding RNAs, are important regulators of gene expression that exert their function through post-transcriptional silencing of complementary mRNA targets. According to recent transcriptomic analyses, certain miRNA species are expressed in all retinal cell types, while others are cell type-specific. As miRNAs play important roles in homeostasis, cellular function, and survival of differentiated retinal cell types, their dysregulation is associated with retinal degenerative diseases. Thus, advancing our understanding of the genetic networks modulated by miRNAs is central to harnessing their potential as therapeutic agents to overcome visual impairment. In this review, we summarize the role of distinct miRNAs in specific retinal cell types, the current knowledge on their implication in inherited retinal disorders, and their potential as therapeutic agents.

Pluripotent Stem Cells as Models of Retina Development.

The ability of pluripotent stem cells (PSCs) to differentiate into retinal tissue has led to many attempts to direct this process to yield specific retinal cell types. The ability to do so would greatly impact both the study of normal retina development in model systems that can be precisely controlled and the generation of a homogeneous population of cells optimized for transplantation in cell replacement therapy. Thus far, many reviews have focused on the translational potential of PSC retinal studies. Here, we focus on the former by summarizing the advances and reflecting on the current limitations to using in vitro differentiation of PSCs into retinal cells and organoids to model in vivo retinal development, with a specific emphasis on photoreceptors. We discuss the versatility of PSC retinal differentiation systems in investigating specific developmental time points that are difficult to assess with classic developmental model systems as well as the potential for efficient screening of factors involved in regulating photoreceptor differentiation. PSCs can be used in conjunction with existing model systems to contribute to the understanding of retina and photoreceptor development, which in turn can enhance the success of using stem cells in translational studies.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method's versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in Müller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery—intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

The condition medium of mesenchymal stem cells promotes proliferation, adhesion and neuronal differentiation of retinal progenitor cells.

Retinal progenitor cell is a promising candidate in the treatment of retinal pigmentosa diseases. The limiting factors of stem cell transplantation are the proliferation and differentiation capacities of hRPCs, which may be governed by culture conditions. Previous studies have proved that the secretome of human Umbilical Cord Mesenchymal stem cells (hUCMSCs) and human Adipose derived stem cells (hADSCs), including more active cytokines and neurotrophic factors, have the paracrine potential of enhancing proliferation and differentiation in several cell types. The aim of this study was to investigate whether hRPCs could effectively proliferate, adhere and differentiate towards specific retinal cell types by treating with the condition medium (CM) of hUCMSCs (hUCMSCCM) or hADSCs (hADSCCM). Here, we show that hUCMSCCM or hADSCCM enhances the proliferation rate of the S and G2 phase cells, with an upregulation of Ki67 expression. Moreover, the upregulation expression of NF, Recoverin and Rhodopsin indicates that specialized retinal cells including ganglion cells and photoreceptors are favored over hRPCs differentiation due to hUCMSCCM or hADSCCM. Under FBS induced differentiation conditions, hRPCs treated with hUCMSCCM or hADSCCM increase the expression of retinal neuron and photoreceptor specific markers. These results suggest that hUCMSCCM and hADSCCM can stimulate the hRPC proliferation, promote its adherence and support hRPC neuronal and photoreceptor differentiation. These findings may provide a new strategy to improve the viability of hRPCs and photoreceptor differentiation capacities.

Bright Light Suppresses Form-Deprivation Myopia Development With Activation of Dopamine D1 Receptor Signaling in the ON Pathway in Retina.

To determine whether dopamine receptor D1 (D1R) signaling pathway activation by bright light (BL) in specific retinal neuronal cell types contributes to inhibiting form-deprivation myopia (FDM) in mice. Mice (3-weeks old) were raised under either normal light (NL: 100-200 lux) or BL (2500-5000 lux) conditions with or without form deprivation. Refraction changes were evaluated with an eccentric infrared photorefractor, and ocular axial components with optical coherence tomography. The D1R antagonist, SCH39166, was intraperitoneally injected daily to evaluate if BL mediates declines in FDM development through D1R activation. Six different biomarkers of retinal neuronal types delineated differential distribution of D1R expression. c-Fos and phosphorylated tyrosine hydroxylase (p-TH) immunofluorescent staining evaluated D1R receptor activation and dopamine synthesis, respectively. Bright light exposure for 4 weeks (6 hours per day) inhibited FDM development by reducing ocular elongation and shifting refraction toward hyperopia compared with changes occurring in NL. SCH39166 injections completely reversed the inhibitory effects of BL on both refraction and ocular elongation. Bright light increased the number of cells expressing p-TH and c-fos. Increases in c-fos+ cells occurred mainly in D1R+ bipolar cells (BCs), especially D1R+ ON-BCs. Bright light increases D1R activity in the BCs of the ON pathway, which is associated with less myopic shift and ocular elongation than those occurring in NL. These declines suggest that increased D1R activity in the ON pathway contributes to the BL suppression of FDM development in mice.

Regulation of Brn3b by DLX1 and DLX2 is required for retinal ganglion cell differentiation in the vertebrate retina.

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo. We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.

Gene Therapy and Stem Cell Transplantation in Retinal Disease: The New Frontier

Gene and cell therapies have the potential to prevent, halt, or reverse diseases of the retina in patients with currently incurable blinding conditions. Over the past 2 decades, major advances in our understanding of the pathobiologic basis of retinal diseases, coupled with growth of gene transfer and cell transplantation biotechnologies, have created optimism that previously blinding retinal conditions may be treatable. It is now possible to deliver cloned genes safely and stably to specific retinal cell types in humans. Preliminary results testing gene augmentation strategies in human recessive diseases suggest promising safety and efficacy profiles, including improved visual function outcomes over extended periods. Additional gene-based strategies under development include approaches to autosomal dominant disease ("gain of function"), attempts to deliver genes encoding therapeutic proteins with proven mechanisms of action interfering with specific disease pathways, and approaches that could be used to render retinal cells other than atrophied photoreceptors light sensitive. In the programs that are the furthest along-pivotal regulatory safety and efficacy trials studying individuals with retinal degeneration resulting from RPE65 mutations-initial results reveal a robust safety profile and clinically significant improvements in visual function, thereby making this program a frontrunner for the first approved gene therapy product in the United States. Similar to gene therapy, progress in regenerative or stem cell-based transplantation strategies has been substantial. It is now possible to deliver safely stem cell-derived, terminally differentiated, biologically and genetically defined retinal pigment epithelium (RPE) to the diseased human eye. Although demonstration of clinical efficacy is still well behind the gene therapy field, multiple programs investigating regenerative strategies in RPE disease are beginning to enroll subjects, and initial results suggest possible signs of efficacy. Stem cells capable of becoming other retinal cell types, such as photoreceptors, are on the cusp of clinical trials. Stem cell-derived transplants can be delivered to precise target locations in the eye, and their ability to ameliorate, reverse, regenerate, or neuroprotect against disease processes can be assessed. Results from these studies will provide foundational knowledge that may lead to clinically significant therapies for currently untreatable retinal disease.

Selective Vulnerability of Specific Retinal Ganglion Cell Types and Synapses after Transient Ocular Hypertension.

Key issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs. Among the α-like RGCs studied, αOFF-transient RGCs exhibited higher rates of cell death, with corresponding reductions in dendritic area, dendritic complexity, and synapse density. Functionally, OFF-transient RGCs displayed decreases in spontaneous activity and receptive field size. In contrast, neither αOFF-sustained nor αON-sustained RGCs displayed decreases in light responses, although they did exhibit a decrease in excitatory postsynaptic sites, suggesting that synapse loss may be one of the earliest signs of degeneration. Interestingly, presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer, corroborating the hypothesis that RGCs with dendrites stratifying in the OFF sublamina may be damaged early. Indeed, OFF arbors of ON-OFF RGCs lose complexity more rapidly than ON arbors. Our results reveal type-specific differences in RGC responses to injury with a selective vulnerability of αOFF-transient RGCs, and furthermore, an increased susceptibility of synapses in the OFF sublamina. The selective vulnerability of specific RGC types offers new avenues for the design of more sensitive functional tests and targeted neuroprotection. Conflicting reports regarding the selective vulnerability of specific retinal ganglion cell (RGC) types in glaucoma exist. We examine, for the first time, the effects of transient intraocular pressure elevation on the structure and function of various RGC types. Among the α-like RGCs studied, αOFF-transient RGCs are the most vulnerable to transient transient intraocular pressure elevation as measured by rates of cell death, morphologic alterations in dendrites and synapses, and physiological dysfunction. Specifically, we found that presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer. Our results suggest selective vulnerability both of specific types of RGCs and of specific inner plexiform layer sublaminae, opening new avenues for identifying novel diagnostic and treatment targets in glaucoma.