Donald J. Zack

Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.

Gonçalo Abecasis and colleagues report a large-scale meta-analysis of genome-wide association studies for age-related macular degeneration (AMD), including over 17,100 advanced cases and 60,000 controls. They identify seven loci newly associated with AMD and report pathway analysis that shows enrichment in the complement system and atherosclerosis signaling. Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10−8. These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10−8 for the first time, near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8, TGFBR1, RAD51B, ADAMTS9 and B3GALTL. A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

To investigate whether retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis.Chronic elevated eye pressure was produced in 20 monkey eyes, and the optic nerve was transected unilaterally in the orbit of 10 monkeys and 14 rabbits. Sixteen monkey and 14 rabbit eyes were studied as normal controls. Analytic methods included light and electron microscopy, histochemistry for DNA fragmentation (TUNEL method), and DNA electrophoresis in agarose gels.Dying ganglion cells in the experimental retinas exhibited morphologic features of apoptosis, including chromatin condensation and formation of apoptotic bodies. Cells with a positive reaction for DNA fragmentation were observed in eyes subjected to axotomy and experimental glaucoma but were only rarely encountered in control eyes. No evidence of internucleosomal fragmentation was detected electrophoretically, possibly because of the small proportion of cells that were dying at any given time.Some retinal ganglion cells injured by glaucoma and by axotomy die by apoptosis.

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10(-75)), ARMS2 (P < 10(-59)), C2/CFB (P < 10(-20)), C3 (P < 10(-9)), and CFI (P < 10(-6)). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 x 10(-11)), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 x 10(-7); CETP, P = 7.4 x 10(-7)) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c-associated alleles near LPL (P = 3.0 x 10(-3)) and ABCA1 (P = 5.6 x 10(-4)). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

To determine whether retinal ganglion cell death in primary open-angle glaucoma occurs by apoptosis.Eighteen eyes of 17 subjects with documented primary open-angle glaucoma were compared with 21 control eyes that were group matched for age, race, and sex. Staging of glaucoma severity was performed by histologic optic nerve evaluation. Fixed, paraffin-embedded retinal sections were assayed by the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (UTP)-biotin nick end-labeling) method to detect the internucleosomal DNA fragmentation that is characteristic of apoptosis.A positive TUNEL reaction was observed among ganglion layer cells in 10 of 18 cases with glaucoma, compared with 1 of 11 control cases without confounding systemic disease (5 control eyes were excluded owing to artifactual staining and 4 eyes had confounding systemic disease). Sections containing more than 250,000 cells in the ganglion cell layer were examined in cases and controls. The frequency of TUNEL-positive cells in the ganglion cell layer in cases with glaucoma was 1.76 per 10,000, or 15.2 times greater than the control frequency from individuals without confounding disease (P < .001; 95% CI, 2.46-623). Eyes without glaucoma from subjects with diabetes and amyotrophic lateral sclerosis showed more positive cells than other controls.Apoptosis seems to be a mechanism of cell death in human eyes with primary open-angle glaucoma.

Advanced age-related macular degeneration (AMD) is the leading cause of late onset blindness. We present results of a genome-wide association study of 979 advanced AMD cases and 1,709 controls using the Affymetrix 6.0 platform with replication in seven additional cohorts (totaling 5,789 unrelated cases and 4,234 unrelated controls). We also present a comprehensive analysis of copy-number variations and polymorphisms for AMD. Our discovery data implicated the association between AMD and a variant in the hepatic lipase gene (LIPC) in the high-density lipoprotein cholesterol (HDL) pathway (discovery P = 4.53e-05 for rs493258). Our LIPC association was strongest for a functional promoter variant, rs10468017, (P = 1.34e-08), that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD. The association we found with LIPC was corroborated by the Michigan/Penn/Mayo genome-wide association study; the locus near the tissue inhibitor of metalloproteinase 3 was corroborated by our replication cohort for rs9621532 with P = 3.71e-09. We observed weaker associations with other HDL loci (ABCA1, P = 9.73e-04; cholesterylester transfer protein, P = 1.41e-03; FADS1-3, P = 2.69e-02). Based on a lack of consistent association between HDL increasing alleles and AMD risk, the LIPC association may not be the result of an effect on HDL levels, but it could represent a pleiotropic effect of the same functional component. Results implicate different biologic pathways than previously reported and provide new avenues for prevention and treatment of AMD.

Deletion of sequences 5′ of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a β-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5′ of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5′ region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Understanding how genes are expressed and regulated in different tissues is a fundamental and challenging question. However, most of currently available biological databases do not focus on tissue-specific gene regulation.The recent development of computational methods for tissue-specific combinational gene regulation, based on transcription factor binding sites, enables us to perform a large-scale analysis of tissue-specific gene regulation in human tissues. The results are stored in a web database called TiGER (Tissue-specific Gene Expression and Regulation). The database contains three types of data including tissue-specific gene expression profiles, combinatorial gene regulations, and cis-regulatory module (CRM) detections. At present the database contains expression profiles for 19,526 UniGene genes, combinatorial regulations for 7,341 transcription factor pairs and 6,232 putative CRMs for 2,130 RefSeq genes.We have developed and made publicly available a database, TiGER, which summarizes and provides large scale data sets for tissue-specific gene expression and regulation in a variety of human tissues. This resource is available at 1.

To develop a modified adenoassociated viral (AAV) vector capable of efficient transfection of retinal ganglion cells (RGCs) and to test the hypothesis that use of this vector to express brain-derived neurotrophic factor (BDNF) could be protective in experimental glaucoma.Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n = 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n = 30), or AAV-green fluorescent protein (GFP)-WPRE (n = 33). Two weeks later, experimental glaucoma was induced in the injected eye by laser application to the trabecular meshwork. Survival of RGCs was estimated by counting axons in optic nerve cross sections after 4 weeks of glaucoma. Transgene expression was assessed by immunohistochemistry, Western blot analysis, and direct visualization of GFP.The density of GFP-positive cells in retinal wholemounts was 1,828 +/- 299 cells/mm(2) (72,273 +/- 11,814 cells/retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% +/- 27.1% in the saline-treated group (n = 25) and 52.3% +/- 24.2% in the AAV-GFP-WPRE group (n = 30), but only 32.3% +/- 23.0% in the AAV-BDNF-WPRE group (n = 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P = 0.002, t-test) or saline (P = 0.006, t-test).Overexpression of the BDNF gene protects RGC as estimated by axon counts in a rat glaucoma model, further supporting the potential feasibility of neurotrophic therapy as a complement to the lowering of IOP in the treatment of glaucoma.

Two common variants in the gene encoding complement factor H (CFH), the Y402H substitution (rs1061170, c.1204C>T)(1-4) and the intronic rs1410996 SNP(5,6), explain 17% of age-related macular degeneration (AMD) liability. However, proof for the involvement of CFH, as opposed to a neighboring transcript, and knowledge of the potential mechanism of susceptibility alleles are lacking. Assuming that rare functional variants might provide mechanistic insights, we used genotype data and high-throughput sequencing to discover a rare, high-risk CFH haplotype with a c.3628C>T mutation that resulted in an R1210C substitution. This allele has been implicated previously in atypical hemolytic uremic syndrome, and it abrogates C-terminal ligand binding(7,8). Genotyping R1210C in 2,423 AMD cases and 1,122 controls demonstrated high penetrance (present in 40 cases versus 1 control, P = 7.0 × 10(-6)) and an association with a 6-year-earlier onset of disease (P = 2.3 × 10(-6)). This result suggests that loss-of-function alleles at CFH are likely to drive AMD risk. This finding represents one of the first instances in which a common complex disease variant has led to the discovery of a rare penetrant mutation.

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63-0.75], p = 1.86×10⁻¹⁸), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21-1.43], p = 3.87×10⁻¹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50-0.67], p = 1.17×10⁻¹²) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53-0.72], p = 8.88×10⁻¹⁰). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41-0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54-1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.

Despite significant progress in the identification of genetic loci for age-related macular degeneration (AMD), not all of the heritability has been explained. To identify variants which contribute to the remaining genetic susceptibility, we performed the largest meta-analysis of genome-wide association studies to date for advanced AMD. We imputed 6 036 699 single-nucleotide polymorphisms with the 1000 Genomes Project reference genotypes on 2594 cases and 4134 controls with follow-up replication of top signals in 5640 cases and 52 174 controls. We identified two new common susceptibility alleles, rs1999930 on 6q21-q22.3 near FRK/COL10A1 [odds ratio (OR) 0.87; P = 1.1 × 10−8] and rs4711751 on 6p12 near VEGFA (OR 1.15; P = 8.7 × 10−9). In addition to the two novel loci, 10 previously reported loci in ARMS2/HTRA1 (rs10490924), CFH (rs1061170, and rs1410996), CFB (rs641153), C3 (rs2230199), C2 (rs9332739), CFI (rs10033900), LIPC (rs10468017), TIMP3 (rs9621532) and CETP (rs3764261) were confirmed with genome-wide significant signals in this large study. Loci in the recently reported genes ABCA1 and COL8A1 were also detected with suggestive evidence of association with advanced AMD. The novel variants identified in this study suggest that angiogenesis (VEGFA) and extracellular collagen matrix (FRK/COL10A1) pathways contribute to the development of advanced AMD.

Numerous protocols have been described that produce neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. While nearly all such methods yield at least partial segments of highly mature-appearing retinal structure, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to better understand the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging, and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. To identify new susceptibility loci, we performed meta-analysis on genome-wide association study (GWAS) results from eight independent studies from the United States (3,853 cases and 33,480 controls) and investigated the most significantly associated SNPs in two Australian studies (1,252 cases and 2,592 controls), three European studies (875 cases and 4,107 controls) and a Singaporean Chinese study (1,037 cases and 2,543 controls). A meta-analysis of the top SNPs identified three new associated loci: rs35934224[T] in TXNRD2 (odds ratio (OR) = 0.78, P = 4.05 × 10(-11)) encoding a mitochondrial protein required for redox homeostasis; rs7137828[T] in ATXN2 (OR = 1.17, P = 8.73 × 10(-10)); and rs2745572[A] upstream of FOXC1 (OR = 1.17, P = 1.76 × 10(-10)). Using RT-PCR and immunohistochemistry, we show TXNRD2 and ATXN2 expression in retinal ganglion cells and the optic nerve head. These results identify new pathways underlying POAG susceptibility and suggest new targets for preventative therapies.

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

Primary open-angle glaucoma (POAG), is a heritable common cause of blindness world-wide. To identify risk loci, we conduct a large multi-ethnic meta-analysis of genome-wide association studies on a total of 34,179 cases and 349,321 controls, identifying 44 previously unreported risk loci and confirming 83 loci that were previously known. The majority of loci have broadly consistent effects across European, Asian and African ancestries. Cross-ancestry data improve fine-mapping of causal variants for several loci. Integration of multiple lines of genetic evidence support the functional relevance of the identified POAG risk loci and highlight potential contributions of several genes to POAG pathogenesis, including SVEP1, RERE, VCAM1, ZNF638, CLIC5, SLC2A12, YAP1, MXRA5, and SMAD6. Several drug compounds targeting POAG risk genes may be potential glaucoma therapeutic candidates.

Thyroid hormone in color vision development Cone photoreceptors in the eye enable color vision, responding to different wavelengths of light according to what opsin pigments they express. Eldred et al. studied organoids that recapitulate the development of the human retina and found that differentiation of cone cells into their tuned subtypes was regulated by thyroid hormone. Cones expressing short-wavelength (S) opsin developed first, and cones expressing long- and medium-wavelength (L/M) opsin developed later. The switch toward development of L/M cones depended on thyroid hormone signaling through the nuclear thyroid hormone receptor. Science , this issue p. eaau6348

The development of single-cell RNA sequencing (scRNA-seq) has allowed high-resolution analysis of cell-type diversity and transcriptional networks controlling cell-fate specification. To identify the transcriptional networks governing human retinal development, we performed scRNA-seq analysis on 16 time points from developing retina as well as four early stages of retinal organoid differentiation. We identified evolutionarily conserved patterns of gene expression during retinal progenitor maturation and specification of all seven major retinal cell types. Furthermore, we identified gene-expression differences between developing macula and periphery and between distinct populations of horizontal cells. We also identified species-specific patterns of gene expression during human and mouse retinal development. Finally, we identified an unexpected role for ATOH7 expression in regulation of photoreceptor specification during late retinogenesis. These results provide a roadmap to future studies of human retinal development and may help guide the design of cell-based therapies for treating retinal dystrophies.

Inherited retinal degenerations (IRDs) represent a diverse group of progressive, visually debilitating diseases that can lead to blindness in which mutations in genes that are critical to retinal function lead to progressive photoreceptor cell death and associated vision loss. IRDs are genetically heterogeneous, with over 260 disease genes identified to date.1 The development of treatments and cures to modify the rate of disease progression has been limited to date, with some success of neurotrophic factor therapy and gene therapies reported from clinical trials.2–11 The best example of treatment success is gene augmentation therapy for IRD caused by mutations in the RPE65 gene, which recently received US Food and Drug Administration (FDA) approval, which in fact represented the first FDA-approved gene therapy (GT) for any genetically inherited disease.4–9 Recent developments in the IRD field have advanced understanding of the mechanisms responsible for vision loss, creating new opportunities to intervene in the course of disease by developing new therapeutic approaches. In 2013, a Delphi-style gathering of IRD experts led to the identification, by consensus, of top priorities to advance therapeutic efforts for IRDs, including the need for systematic genotyping, improved standardization of visual function testing, development of more rigorous and widespread data collection protocols, and increased data sharing.12 This document summarizes more recent advances in the IRD field and outlines specific knowledge gaps. These knowledge gaps present opportunities for further investigation to enable development of therapies that may slow down or prevent vision loss, or restore vision, in affected patients. Atrophic age-related macular degeneration (AMD) is included among the target inherited retinal diseases of interest because first, understanding AMD may contribute to understanding of inherited macular diseases, and second, understanding of the genetics and mechanism of inherited macular degenerations may contribute to understanding of AMD.

Nanoparticle-based mRNA therapeutics hold great promise, but cellular internalization and endosomal escape remain key barriers for cytosolic delivery. We developed a dual nanoparticle uptake and endosomal disruption assay using high-throughput and high-content image-based screening. Using a genetically encoded Galectin 8 fluorescent fusion protein sensor, endosomal disruption could be detected via sensor clustering on damaged endosomal membranes. Simultaneously, nucleic acid endocytosis was quantified using fluorescently tagged mRNA. We used an array of biodegradable poly(beta-amino ester)s as well as Lipofectamine and PEI to demonstrate that this assay has higher predictive capacity for mRNA delivery compared to conventional polymer and nanoparticle physiochemical characteristics. Top nanoparticle formulations enabled safe and efficacious mRNA expression in multiple tissues following intravenous injection, demonstrating that the in vitro screening method is also predictive of in vivo performance. Efficacious nonviral systemic delivery of mRNA with biodegradable particles opens up new avenues for genetic medicine and human health.

Recent advances in human stem cell-derived brain organoids promise to replicate critical molecular and cellular aspects of learning and memory and possibly aspects of cognition in vitro . Coining the term “organoid intelligence” (OI) to encompass these developments, we present a collaborative program to implement the vision of a multidisciplinary field of OI. This aims to establish OI as a form of genuine biological computing that harnesses brain organoids using scientific and bioengineering advances in an ethically responsible manner. Standardized, 3D, myelinated brain organoids can now be produced with high cell density and enriched levels of glial cells and gene expression critical for learning. Integrated microfluidic perfusion systems can support scalable and durable culturing, and spatiotemporal chemical signaling. Novel 3D microelectrode arrays permit high-resolution spatiotemporal electrophysiological signaling and recording to explore the capacity of brain organoids to recapitulate the molecular mechanisms of learning and memory formation and, ultimately, their computational potential. Technologies that could enable novel biocomputing models via stimulus-response training and organoid-computer interfaces are in development. We envisage complex, networked interfaces whereby brain organoids are connected with real-world sensors and output devices, and ultimately with each other and with sensory organ organoids (e.g. retinal organoids), and are trained using biofeedback, big-data warehousing, and machine learning methods. In parallel, we emphasize an embedded ethics approach to analyze the ethical aspects raised by OI research in an iterative, collaborative manner involving all relevant stakeholders. The many possible applications of this research urge the strategic development of OI as a scientific discipline. We anticipate OI-based biocomputing systems to allow faster decision-making, continuous learning during tasks, and greater energy and data efficiency. Furthermore, the development of “intelligence-in-a-dish” could help elucidate the pathophysiology of devastating developmental and degenerative diseases (such as dementia), potentially aiding the identification of novel therapeutic approaches to address major global unmet needs.

In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.

In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys.Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus.With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes.Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.

To determine whether acute experimental glaucoma in rats obstructs retrograde transport of brain-derived neurotrophic factor (BDNF) to retinal ganglion cells (RGCs).Forty rats had unilateral injection of either (125)I-BDNF (20 animals) or a mixture of (125)I-BDNF and 100-fold excess nonradiolabeled BDNF (20 animals). In each group of 20 animals, eyes contralateral to injection had either normal intraocular pressure (IOP; 10 animals) or IOP elevated to 25 mm Hg below the systolic blood pressure of the eye (10 animals). In each group of 20 rats, ipsilateral eyes had IOP set at systolic blood pressure (4 eyes), had optic nerve transection (10 eyes), or had normal IOP (6 eyes). Six hours after injection, animals were killed and tissues were fixed, embedded, and sectioned for autoradiography. Grain counts were performed over retina and optic nerve using automated image analysis.IOP elevation to 25 mm Hg below systolic blood pressure (perfusion pressure [PP] 25) decreased median retinal nerve fiber layer (NFL) grains by 38% compared with controls (P: < 0.001). Competition by cold BDNF reduced NFL grains by 28% (P: = 0.013). Considering only the radioactivity representing specific retrograde transport of BDNF, IOP elevation to PP25 reduced transport by 74%, whereas elevation to PP0 (equaling systolic blood pressure) reduced specific transport by 83%.BDNF is transported retrogradely from the superior colliculus in adult rats, and this transport is substantially inhibited by acute IOP elevation. Deprivation of BDNF among RGCs may contribute to neuron loss in glaucoma.

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.

Age-related macular degeneration is the most common cause of irreversible visual impairment in the developed world. Advanced age-related macular degeneration consists of geographic atrophy and choroidal neovascularization. The specific genetic variants that predispose patients to geographic atrophy are largely unknown.We tested for an association between the functional toll-like receptor 3 gene (TLR3) variant rs3775291 (involving the substitution of phenylalanine for leucine at amino acid 412) and age-related macular degeneration in Americans of European descent. We also tested for the effect of TLR3 Leu and Phe variants on the viability of human retinal pigment epithelial cells in vitro and on apoptosis of retinal pigment epithelial cells from wild-type mice and Tlr3-knockout (Tlr3(-/-)) mice.The Phe variant (encoded by the T allele at rs3775291) was associated with protection against geographic atrophy (P=0.005). This association was replicated in two independent case-control series of geographic atrophy (P=5.43x10(-4) and P=0.002). No association was found between TLR3 variants and choroidal neovascularization. A prototypic TLR3 ligand induced apoptosis in a greater fraction of human retinal pigment epithelial cells with the Leu-Leu genotype than those with the Leu-Phe genotype and in a greater fraction of wild-type mice than Tlr3(-/-) mice.The TLR3 412Phe variant confers protection against geographic atrophy, probably by suppressing the death of retinal pigment epithelial cells. Since double-stranded RNA (dsRNA) can activate TLR3-mediated apoptosis, our results suggest a role of viral dsRNA in the development of geographic atrophy and point to the potential toxic effects of short-interfering-RNA therapies in the eye.

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function. Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function. rhodopsin proximal promoter region basic leucine zipper glutathioneS-transferase bovine CRX homeodomain Gene activation is a stringently controlled process, involving combinatorial and cooperative action of multiple regulatory proteins with promoter and enhancer DNA elements (1Ptashne M. Gann A. Nature. 1997; 386: 569-577Crossref PubMed Scopus (945) Google Scholar, 2Kuras L. Struhl K. Nature. 1999; 389: 609-613Crossref Scopus (399) Google Scholar, 3Li X.-L. Virbasius A. Zhu X. Green M.R. Nature. 1999; 389: 605-609Crossref Scopus (206) Google Scholar, 4Goodrich J.A. Cutler G. Tijan R. Cell. 1996; 84: 825-830Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Leftsin J.A. Yamamoto K.R. Nature. 1998; 392: 885-888Crossref PubMed Scopus (439) Google Scholar, 6Blackwood E.M. Kadonaga J.T. Science. 1998; 281: 60-63Crossref PubMed Scopus (619) Google Scholar). Recent studies, including reconstitution experiments, have suggested that target specificity and transcriptional synergy are achieved by specific and precise interactions among various activator proteins during the assembly of higher order nucleoprotein complexes, including the “enhanceosome” (7Tijan R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 8Grosschedl R. Curr. Opin. Cell Biol. 1995; 7: 362-370Crossref PubMed Scopus (151) Google Scholar, 9Kim T.K. Maniatis T. Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 10Carey M. Cell. 1998; 92: 5-8Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 11Merika M. Williams A.J. Chen G. Collins T. Thanos D. Mol. Cell. 1998; 1: 277-287Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The elucidation of these protein-protein and protein-DNA interactions is critical to our understanding of the mechanisms of cell type- and tissue-specific gene expression. Generation of multiple neuronal cell types during retinal development is an evolutionarily conserved biological process, which offers a convenient model system to investigate tissue-specific gene regulation. More than 30 transcription factors representing several classes of DNA-binding proteins are expressed in developing and mature mammalian retina; nevertheless, the precise function of a majority of these proteins remains to be elucidated (12Freund C. Horsford D.J. McInnes R.R. Hum. Mol. Gen. 1996; 5: 1471-1488Crossref PubMed Google Scholar, 13Mathers P.H. Grinsberg A. Makon K.A. Jamrich M. Nature. 1997; 387: 603-607Crossref PubMed Scopus (584) Google Scholar, 14Cepko C.L. Austin C.P. Yang X. Alexiades M. Ezzeddine D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 589-595Crossref PubMed Scopus (842) Google Scholar). Rhodopsin, the G-protein-coupled light receptor, is expressed specifically in the rod photoreceptors of retina and is a pivotal protein for visual function. Its expression is correlated to rod differentiation and maintained at high levels afterward, throughout life (15Morrow E.M. Furukawa T. Cepko C.L. Trends Cell Biol. 1998; 8: 353-358Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Altered expression of rhodopsin and mutations that affect its function in mature rods result in retinal degeneration (15Morrow E.M. Furukawa T. Cepko C.L. Trends Cell Biol. 1998; 8: 353-358Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Rattner A. Sun H. Nathans J. Annu. Rev. Gen. 1999; 33: 89-131Crossref PubMed Scopus (197) Google Scholar). Regulation of rhodopsin expression is primarily at the level of transcription and is mediated by two distinct regions: a proximal sequence from −176 to +70 bp, which determines photoreceptor specificity (called the rhodopsin proximal promoter region (RPPR)),1 and another more upstream region required for high level expression (called the rhodopsin enhancer region) (17Zack D.J. Bennet J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 18Nathans J. Biochemistry. 1992; 31: 4923-4931Crossref PubMed Scopus (166) Google Scholar, 19Timmers A.M. Newton B.R. Hauswirth W.W. Exp. Eye. Res. 1993; 56: 257-265Crossref PubMed Scopus (32) Google Scholar, 20Treisman J.E. Morabito M.A. Barnstable C.J. Mol. Cell. Biol. 1988; 8: 1570-1579Crossref PubMed Scopus (93) Google Scholar, 21Lem J. Applebury M.L. Falk J.D. Flannery J.G. Simon M.I. Neuron. 1991; 6: 201-210Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 22Nie Z. Chen S. Kumar R. Zack D.J. J. Biol. Chem. 1996; 271: 2667-2675Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). A number of DNA sequence elements that bind to nuclear proteins have been identified within RPPR; these include Ret-1/PCE-1 (23Morabito M.A., Yu, X. Barnstable C.J. J. Biol. Chem. 1991; 266: 9667-9672Abstract Full Text PDF PubMed Google Scholar, 24Kikuchi T. Raju K. Breitman M.L. Shinohara T. Mol. Cell. Biol. 1993; 13: 4400-4408Crossref PubMed Scopus (105) Google Scholar), BAT-1 (25DesJardin L.E. Hauswirth W.W. Invest. Ophthalmol. Vis. Sci. 1996; 37: 154-165PubMed Google Scholar), eopsin-1 (26Ahmad I. Dev. Brain. Res. 1995; 90: 184-189Crossref PubMed Scopus (51) Google Scholar), Ret-4 (27Chen S. Zack D.J. J. Biol. Chem. 1996; 271: 28549-28557Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and NRE (28Rehemtulla A. Warwar R. Kumar R. Ji X. Zack D.J. Swaroop A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 191-195Crossref PubMed Scopus (186) Google Scholar) (Fig. 1). NRL, a basic leucine zipper (bZIP) protein of the Maf subfamily (29Swaroop A. Xu J. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (269) Google Scholar), was the first transcription factor shown to bind to NRE in the RPPR region and transactivate the rhodopsin promoter in cultured cells (28Rehemtulla A. Warwar R. Kumar R. Ji X. Zack D.J. Swaroop A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 191-195Crossref PubMed Scopus (186) Google Scholar, 30Kumar R. Chen S. Scheurer D. Wang Q. Duh E. Sung C. Rehemtulla A. Swaroop A. Adler R. Zack D.J. J. Biol. Chem. 1996; 271: 29612-29618Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Soon thereafter, CRX, a photoreceptor-specific paired-like homeodomain protein, was identified as the Ret-4 and BAT-1 binding protein by yeast one-hybrid screening and shown to activate the promoters of rhodopsin and other retinal genes (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). NRL and CRX demonstrated transcriptional synergy in rhodopsin promoter activation when transfected together in cultured cells (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Recently, the Ret-1/PCE-1 element was shown to bind two other homeodomain proteins, Erx (32Martinez J.A. Barnstable C.J. Biochem. Biophys. Res. Commun. 1998; 250: 175-180Crossref PubMed Scopus (27) Google Scholar) and Rx (33Kimura A. Singh D. Wawrousek E.F. Kikuchi M. Nakamura M. Shinohara T. J. Biol. Chem. 2000; 275: 1152-1160Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). During embryonic development in mice, Nrl transcripts are detected in all postmitotic neurons and lens; however, its expression becomes restricted primarily to the retinal photoreceptors in the adult (34Liu Q. Ji X. Breitman M.L. Hitchcock P.F. Swaroop A. Oncogene. 1996; 12: 207-211PubMed Google Scholar). CRX is expressed specifically in photoreceptors and pinealocytes and plays a significant role during photoreceptor differentiation (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 35Furukawa T. Morrow E.M. Cepko C.L. Cell. 1997; 91: 531-541Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar,36Furukawa T. Morrow E.M. Li T. Davis F.C. Cepko C.L. Nat. Genet. 1999; 23: 466-470Crossref PubMed Scopus (446) Google Scholar). Consistent with their role in rhodopsin regulation, detection of NRL and CRX transcripts precedes rhodopsin expression during rod development in mammals. In addition, mutations in the humanCRX and NRL genes have been identified in retinopathies, and these mutations result in altered transcriptional synergy in rhodopsin promoter activation assays (37Freund C.L. Gregory-Evans C.Y. Furukawa T. Papaioannou M. Looser J. Ploder L. Bellingham J. Ng D. Herbrick J.-A.S. Duncan A. Scherer S.W. Tsui L.-C. Loutradis-Anagnostou A. Jacobson S.G. Cepko C.L. Bhattacharya S.S. McInnes R.R. Cell. 1997; 91: 543-553Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 38Swain P.K. Chen S. Wang Q.-L. Affatigato L.M. Coats C.L. Brady K.D. Fishman G.A. Jacobson S.G. Swaroop A. Stone E. Sieving P.A. Zack D.J. Neuron. 1997; 19: 1329-1336Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 39Freund C.L. Wang Q.-L. Chen S. Muskat B.L. Wiles C.D. Sheffield V.C. Jacobson S.G. McInnes R.R Zack D.J. Stone E.M. Nat. Genet. 1998; 18: 311-312Crossref PubMed Scopus (253) Google Scholar, 40Swaroop A. Wang Q.L. Wu W. Cook J. Coats C. Xu S. Chen S. Zack D.J. Sieving P.A. Hum. Mol. Genet. 1999; 8: 299-305Crossref PubMed Scopus (156) Google Scholar, 41Bessant D.A.R. Payne A.M. Mitton K.P. Wang Q.-L. Swain P.K. Plant C. Bird A.C. Zack D.J. Swaroop A. Bhattacharya S.S. Nat. Genet. 1999; 21: 355-356Crossref PubMed Scopus (152) Google Scholar). Based on these findings, we hypothesized that the transcriptional synergy between NRL and CRX in rhodopsin regulation results from cooperativity in binding to adjacent NRE and Ret-4 (or BAT-1) sites and/or from direct physical interaction, leading to the formation of a stable enhanceosome and/or initiation complex. In this paper, we demonstrate direct interaction between the leucine zipper of NRL and the homeodomain of CRX using the yeast two-hybrid interaction trap andin vitro glutathione S-transferase (GST) pull-down assays. The two-hybrid screening in yeast was carried out according to the published procedure (42Golemis E.A. Gyuris J. Brent R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1996: 20.1.1-20.1.28Google Scholar), with minor modifications. The bait construct was generated by cloning the SacII–PpuMI fragment of the human NRL cDNA (29Swaroop A. Xu J. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (269) Google Scholar) at the PvuII site of the pHybLex/Zeo vector (Invitrogen, Carlsbad, CA). The resulting construct, called pLex-NRL-ZIP, encoded the NRL leucine zipper (NRL-ZIP; amino acids 171–231) fused in frame with the LexA protein and did not display autologous activation of the reporter gene lacZ orHIS3 upon transformation in the yeast L40 strain (MATa his3Δ200 trp1–901 leu2–3112 ade2 LYS2::(4lexAop-HIS3) URA3::(8lexAop-lacZ) GAL4). The following constructs were obtained as part of the “Hybrid Hunter System” (from Invitrogen) and used for positive and negative interaction control experiments: pHybLex/Zeo-Fos2, pHybLex/Zeo-Laminin, and pYESTrp-Jun, which expressed the c-Fos leucine-zipper domain fused to LexA, laminin fused to LexA, and the c-Jun leucine zipper fused to the B42-activation domain (B42-AD), respectively. A bovine retina cDNA library (43Tai A.W. Chuang J.-Z. Bode C. Wolfrum U. Sung C.-H. Cell. 1999; 97: 877-887Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) in pACTII (prey vector with Gal4-activation domain) (a generous gift of Dr. C. H. Sung) was used to isolate interacting clones. Yeast strain L40 was sequentially transformed with pLex-NRL-ZIP and then with 10 μg of DNA from the retina library or purified clones in the prey vector, essentially as described (44Gietz D. Jean A.S. Woods R.A. Shiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Double transformants were selected for presence of the bait and the prey vectors, and possible interactors were selected by growth on the appropriate yeast minimal medium (250 μm zeocin, minus Leu His) and by filter lift assay of β-galactosidase activity. Mixed bait and prey construct DNAs were recovered from double-positive yeast transformants after Zymolase-20T/SDS treatment (ICN Biomedicals, Aurora, OH). The pACTII retina library clones were separated from the bait vector DNA by electroporation into Escherichia coli XL1-Blue bacteria and growth on LB-ampicillin plates. Plasmid DNA, prepared by the alkaline lysis method, was used for sequencing and to test for false positives by retransformation of L40 yeast strains containing pHybLex/Zeo-Laminin and pLex-NRL-ZIP bait vectors. NRL cDNA fragments corresponding to the full-length protein of 237 amino acids and a truncated protein with the bZIP domain (amino acids 110–237; ΔNRL) were cloned in frame with GST in the pGex2TK vector (Amersham Pharmacia Biotech). The bovine CRX (bCRX) cDNA was cloned in pcDNA3.1/HisC mammalian expression vector (Invitrogen, Carlsbad, CA). A series of N- and C-terminal deletions were generated by polymerase chain reaction amplification usingPfu DNA polymerase (Strategene, La Jolla, CA), with the wild-type bCRX and primers corresponding to the appropriate end sequences with added BamHI (5′) or EcoRI (3′) site (see Fig. 5 A). The resulting polymerase chain reaction fragments were digested with BamHI and EcoRI, gel-purified, and subcloned intoBamHI/EcoRI-digested pcDNA 3.1/HisC vector. All deletions are fused in frame with the His6 tag at the N terminus and contain a stop codon at the C terminus. The sequence of each deletion construct was confirmed using a Perkin-Elmer ABI Prism DNA sequencing kit and ABI Prism 310 Genetic Analyzer. The GST, GST-NRL, and GSTΔNRL proteins were produced in E. coli strain BL21, essentially as described (45Artandi S. Calame K. Methods Mol. Genet. 1993; 1: 267-278Google Scholar). Briefly, the transformed bacteria were grown at 37 °C for 4 h (A600∼0.8) and induced with 0.5 mmisopropyl-thio-β-d-galactopyranoside for 3 h at 27 °C. Cells were disrupted by sonication in the lysis buffer (20 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA, and 1× Complete protease inhibitor mixture (Amersham Pharmacia Biotech)). E. coli-expressed proteins were purified using glutathione-Sepharose beads, as suggested by the manufacturer (Amersham Pharmacia Biotech). Protein concentrations were estimated using bicinchoninic acid reagent (Sigma). The GST-CRX-HD fusion protein was expressed and purified from E. coli BL21, as described previously (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). bCRX constructs in pcDNA3.1/HisC vector (0.3 μg of double-stranded DNA) were translated in a 25-μl reaction in the presence of [35S]methionine (>1000 μCi/mmol; Amersham Pharmacia Biotech) using the T7-TNT Quick Coupled Transcription/Translation SystemTM (Promega). For in vitro interaction experiments, 7 μl of the 35S-labeled protein was incubated with glutathione-Sepharose-bound GST, GST-NRL, or GSTΔNRL protein (≥100 μg) in the binding buffer (20 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.2% Nonidet P-40). After 18 h of incubation at 4 °C on a nutator, the beads were washed five times in buffer containing 20 mm Tris-Cl, pH 8.0, 150 mm NaCl and, 0.2% Triton X-100. After the final wash, glutathione-Sepharose-bound proteins were resuspended in 60 μl of 2× SDS sample buffer, boiled for 5 min, and separated by SDS-polyacrylamide gel electrophoresis. To estimate the amount of labeled protein used in binding, 20% of the in vitrotranslated products were also examined on a parallel gel. The radiolabeled proteins were visualized by fluorography after treatment of the gel with AmplifyTM (Amersham Pharmacia Biotech), as described (46Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar). For in vitro translation studies, the NRL cDNA (29Swaroop A. Xu J. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (269) Google Scholar) was subcloned in pcDNA3.1 (Invitrogen). The plasmid construct was linearized by different restriction enzymes that digest at a unique site, and 35S-labeled full-length or truncated NRL proteins were produced using the T7-TNT Quick Coupled Transcription/Translation SystemTM (Promega). As an initial approach to evaluate the possibility of direct interaction between NRL and CRX, the yeast two-hybrid method was employed. We generated a bait vector that produced a fusion protein with LexA and the leucine zipper domain of NRL (NRL-ZIP). This bait construct did not autoactivate the HIS3 and lacZ reporter genes upon transformation in L40 yeast. The NRL-ZIP bait was used for screening a bovine retinal cDNA library in the pACTII vector (43Tai A.W. Chuang J.-Z. Bode C. Wolfrum U. Sung C.-H. Cell. 1999; 97: 877-887Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Twenty-eight yeast double transformants that displayed fast growth on minus His medium were selected for further analysis. Filter lift tests of these His+ clones identified 26 clones that expressed β-galactosidase activity as well. Sequence analysis of the double positive clones revealed that 21 of them had bCRX sequence fused in frame to the Gal-4 activation domain. These clones could be divided into five subsets; four subsets included the 5′ noncoding sequence of bCRX, whereas one began at codon 14 of bCRX (Fig.2). All of the bCRX clones obtained from the screen contained the homeodomain. These clones, with or without the additional sequence from the 5′-untranslated region, were retransformed into L40 yeast. The presence of both the bait vector pLex-NRL-ZIP and bCRX-prey clones was found to be essential for growth on minus His medium (Fig. 3).Figure 3NRL-leucine zipper domain interacts with CRX in the yeast two-hybrid assay. A, the pACTII prey clones obtained from the screening of a bovine retina library were transformed into yeast L40 with and without the bait vector pLex-NRL-ZIP and replica-streaked onto minimal medium (minus Leu His, plus 10 mm aminotriazole) or minimal medium (minus Leu His, plus 250 μm zeocin, 10 mm aminotriazole). Three examples are shown here: Gal4-AD/CRX fusions without the 5′ noncoding sequence (clone CRX-N14), with the 5′ noncoding sequence (clone CRX-A), and a novel clone (KM1698–1). Only L40 double transformants with the bait vector grew on minus His plates.B, interaction of CRX is specific for the NRL-leucine zipper domain. Bait yeast strains L40/pHybLex/Zeo-Laminin and L40/pLex-NRL-ZIP were transformed with the CRX-N14 DNA, and double transformants were streaked onto minimal medium (minus Leu, minus His, plus 250 μm zeocin and 50 mm aminotriazole). The yeast transformants with the laminin bait and CRX prey did not grow, whereas NRL-ZIP and CRX double transformants activated the HIS3reporter gene and grew well. C, LexA-c-Fos (pHybLex/Zeo-Fos2) interacts with B42-AD-c-Jun (pYESTrp-Jun) in L40 yeast under identical assay conditions, whereas double transformants of LexA-laminin (pHybLex/Zeo-Laminin) with B42-AD-c-Jun did not show any interaction. Double transformants with c-Jun/c-Fos grew well on minimal medium (minus Trp, minus His, plus 250 μm zeocin and 50 mm aminotriazole), while those with laminin/c-Fos did not.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Specificity of interaction between NRL-ZIP and bCRX was confirmed by transformation of pACTII-bCRX(N14) with pLex-NRL-ZIP or pHybLex/Zeo-Laminin and streaking the double transformants onto minus His plates containing 50 mm aminotriazole. As shown in Fig. 3 B, only yeast double transformants with pLex-NRL-ZIP grew on minus His plates, while the presence of the laminin bait construct did not result in the activation of the HIS3reporter gene. Under identical assay conditions, double transformants of L40 yeast with the c-Fos (pHybLex/Zeo-Fos2) bait and c-Jun (pYESTrp-Jun) prey constructs showed excellent growth on minus His plates, whereas the double transformants with the laminin bait (pHybLex/Zeo-Laminin) and c-Jun prey did not (Fig. 3 C). To confirm the direct interaction of NRL and CRX, we employed a pull-down assay with GST fusion proteins. Both GST-NRL (expressing GST fused with the full-length NRL protein) and GSTΔNRL (GST fused with the C-terminal 117 amino acids of NRL; primarily the bZIP domain), but not GST alone, were able to interact with the in vitrotranslated full-length CRX protein (Fig.4). This was consistent with the yeast two-hybrid screening, where NRL-ZIP bait was used. The addition of micrococcal nuclease to the GST pull-down reactions (in order to remove the DNA template) did not significantly alter the interaction of CRX with NRL (data not shown). To further define the region of bCRX protein that interacts with NRL, various deletion constructs (shown in Fig.5 A) were used for in vitro translation. In the pull-down assay, full-length (construct bCRX) and truncated proteins containing the HD and the glutamine-rich plus basic region (constructs N34, C160, and C208) demonstrated strong binding to GSTΔNRL (Fig. 5 B). Deletion of the N-terminal 87 residues (construct N88), which include most of the homeodomain, considerably reduced the binding of bCRX to GSTΔNRL, whereas the removal of the N-terminal 111 residues (construct N112, which also removes the Gln region) completely eliminated the binding (Fig.5 B). Deletion of the first 33 amino acids in bCRX (construct N34) did not have any effect on the interaction. Truncated bCRX proteins, which included the homeodomain (constructs HD and C107), consistently showed binding to NRL, although at a reduced level (Fig.5 B). SDS-PAGE analysis of the in vitro translated bCRX proteins revealed a comparable intensity of the labeled protein in binding reactions (Fig. 5 C). Taken together, the data strongly suggest that amino acids 34–88 (i.e. the homeodomain) constitute a region of CRX that is important for interaction with NRL-bZIP. Nonetheless, the binding efficiency was enhanced dramatically when the glutamine-rich plus basic region that follows the HD was included. Together with the yeast two-hybrid experiments, these results suggest that CRX-HD provides the primary interface for physical interaction with the leucine zipper motif of NRL. To define the domain of NRL responsible for interaction with the CRX homeodomain, GST-CRX-HD fusion protein was used for GST pull-down assay of in vitro translated NRL proteins. Full-length NRL and the NRL proteins with C-terminal deletions after amino acids 210 and 190 (NRL210 and NRL190) were prepared by in vitro translation. Full-length NRL displayed strong interaction with GST-CRX-HD by pull-down assay (Fig.6). Removal of the leucine zipper domain (either in part or most of it, constructs NRL210 and NRL190) decreased NRL's binding to CRX-HD (Fig. 6) and its ability to form a homodimer (data not shown). To further ascertain the importance of HD in NRL-CRX interaction, two mutations (R41W and R90W, identified in autosomal dominant cone-rod dystrophy (38Swain P.K. Chen S. Wang Q.-L. Affatigato L.M. Coats C.L. Brady K.D. Fishman G.A. Jacobson S.G. Swaroop A. Stone E. Sieving P.A. Zack D.J. Neuron. 1997; 19: 1329-1336Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) and Leber congenital amaurosis (40Swaroop A. Wang Q.L. Wu W. Cook J. Coats C. Xu S. Chen S. Zack D.J. Sieving P.A. Hum. Mol. Genet. 1999; 8: 299-305Crossref PubMed Scopus (156) Google Scholar), respectively) were incorporated in the CRX expression construct. The mutant CRX proteins, synthesized in vitro, were then used for the pull-down assay with GSTΔNRL. Both HD mutations, R41W and R90W, resulted in reduced binding to NRL-bZIP (Fig.7), consistent with the role of HD as the primary interface for interaction. Using two independent methods, we have provided evidence for direct physical interaction of NRL and CRX, two transcription factors implicated in rhodopsin regulation. The yeast two-hybrid studies show that the interaction is stable and functional within a cellular environment, whereas the GST pull-down experiments demonstrate direct association in vitro. These studies also indicate that the leucine zipper domain of NRL is sufficient for binding to CRX. Yeast two-hybrid experiments with LexA-bait fusions comprising the N-terminal or C-terminal halves of NRL-ZIP also showed interaction with the CRX prey (data not shown), suggesting a broad surface of contacts along the leucine zipper. Interestingly, the x-ray crystal structure of the interaction of the leucine zipper dimer AP1 (c-Fos/c-Jun) with NFAT, a β-scaffold type DNA-binding domain protein, illustrates an interaction surface that extends along the length of the leucine zipper region (47Chen L. Glover J.N.M. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (414) Google Scholar). The bZIP transcription factors have been shown to interact with several other protein domains, and such interaction can either activate or repress expression of downstream target genes (48Yang-Yen H.-F. Chambard J.-C. Sun Y.-L. Smeal T. Schmidt T.J. Drouin J. Karin M. Cell. 1990; 62: 1205-1215Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 49Gavva N.R. Gavva R. Ermekova K. Sudol M. Shen C.-K.J. J. Biol. Chem. 1997; 272: 24105-24108Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 50Yamaguchi Y. Wada T. Suzuki F. Takagi T. Hasegawa J. Handa H. Nucleic Acids Res. 1998; 26: 3854-3861Crossref PubMed Scopus (43) Google Scholar, 51Agarwal S.K. Siradanahalli C.G. Heppner C. Erdos M.R. Collins R.M. Park S.Y. Saggar S. Chandrasekharappa S.C. Collins F.S. Spiegel A.M. Marx S.J. Burns A.L. Cell. 1999; 96: 143-152Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Our studies demonstrate that the bZIP motif is capable of interacting with the homeodomain as well. Although deletions can cause allosteric effects, the GST pull-down studies using CRX-HD with NRL deletion constructs (see Fig. 6) are consistent with this conclusion. While the leucine zipper region is sufficient for interaction with CRX (and specifically CRX-HD), it is probably not the only NRL domain that can interact with CRX. Other regions in NRL might also provide additional contact surfaces for interaction (see Fig.6). 2K. P. Mitton and A. Swaroop, unpublished data. During the yeast two-hybrid screening, we did not identify any leucine zipper proteins. This was surprising, since leucine zippers are shown to heterodimerize with other leucine zipper proteins. It is possible that CRX is the predominant interacting protein, and we did not exhaustively screen the prey library. Nonetheless, we think that the lack of leucine zipper proteins is due to the design of the bait construct. LexA binds to DNA as a homodimer through its N-terminal domain, while dimerization is mediated by its C-terminal domain (52Schnarr M. Oertel-Buchheit P. Kazamaier M. Granger-Schnarr M. Biochemie. 1991; 73: 423-431Crossref PubMed Scopus (91) Google Scholar). The use of NRL-ZIP as LexA fusion bait presents two possible outcomes. If the NRL-ZIP does not homodimerize, then the bait domain will be free to interact and form other potential leucine zipper associations with prey proteins from the retina library. This is illustrated by the fact that LexA-c-Fos bait fusion is free to interact with the B42-AD-c-Jun fusion protein, since the c-Fos leucine zipper does not homodimerize. On the other hand, if the NRL-ZIP forms a stable homodimer, complemented by LexA dimerization, it will not be available to interact with other leucine zipper domains. Nevertheless, prey proteins that interact with the exterior surface of the NRL-ZIP dimer would be picked up in this yeast two-hybrid screening. Analysis of the NRL-ZIP domain sequence reveals that the NRL-NRL homodimer has six potential ionic interactions, which would favor dimerization of similar strength as Fos-Jun association. The strong interaction of NRL with itself in GST pull-down assays strongly supports this possibility (data not shown). The yeast two-hybrid screening results are consistent with the hypothesis that NRL-ZIP bait exists as a homodimer and that the surface with leucine residues in the zipper is unavailable for interaction. We suggest that bCRX interacts with the NRL-ZIP homodimer and that the NRL interaction surface is the outside surface of the dimer (positionsa and d around the helical wheel) (53Glover J.N.M. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (671) Google Scholar). Additional experiments will be necessary to test this hypothesis. Homeodomains bind to DNA, and this is true for CRX as well (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 38Swain P.K. Chen S. Wang Q.-L. Affatigato L.M. Coats C.L. Brady K.D. Fishman G.A. Jacobson S.G. Swaroop A. Stone E. Sieving P.A. Zack D.J. Neuron. 1997; 19: 1329-1336Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar,40Swaroop A. Wang Q.L. Wu W. Cook J. Coats C. Xu S. Chen S. Zack D.J. Sieving P.A. Hum. Mol. Genet. 1999; 8: 299-305Crossref PubMed Scopus (156) Google Scholar). Based on the published structure of the paired homeodomain (54Wilson D.S. Guenther B. Desplan C. Kuriyan J. Cell. 1995; 82: 709-719Abstract Full Text PDF PubMed Scopus (305) Google Scholar), we hypothesize that CRX-HD has three helix motifs: h1, from residues 50–59; h2, residues 69–76; and h3, residues 80–89. Helix-3 should be important for making contacts with DNA. It would then appear that the helices h1 and h2 are more accessible for interaction with NRL, consistent with the yeast two-hybrid screening results and CRX deletion analysis in GST pull-down assays. Both h1 and h2 are rich in ionic and polar amino acids that can make salt-bridge and hydrogen bonds with the NRL interaction surface. The conclusion that CRX-HD is involved in both DNA binding and protein-protein interaction is strengthened by the GST pull-down experiments with mutant CRX proteins (see Fig. 7). The glutamine-rich plus basic region that follows the homeodomain could provide a surface for additional interaction, since it enhanced the binding in pull-down assays. Homeodomain proteins appear to have a broad influence on gene expression, since they bind to a wide range of DNA sequences with similar affinity and this binding can be positively correlated to transcriptional activity (55Desplan C. Theis J. O'Farrell P.H. Cell. 1988; 54: 1081-1090Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 56Carr A. Biggin M.D. EMBO J. 1999; 18: 1598-1608Crossref PubMed Scopus (76) Google Scholar). CRX may have a similar global influence on photoreceptor-specific gene expression, since it can bind to and transactivate from regulatory elements in several photoreceptor-specific genes, including rhodopsin, interphotoreceptor retinoid-binding protein, arrestin, and β-phosphodiesterase (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 57Livesey F.J. Furukawa T. Steffen M.A. Church G.M. Cepko C.L. Curr. Biol. 2000; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). In contrast, nonhomeodomain transcription factors bind to promoters in fewer genes with greater sequence specificity (58Bulger M. Groudine M. Genes Dev. 1999; 13: 2465-2477Crossref PubMed Scopus (375) Google Scholar). Such transcription factors, like NRL, may influence the specificity of gene transcription while homeodomain proteins, such as CRX, may facilitate structural control of larger chromatin regions and help facilitate the effects of other transcription factors upon the enhanceosome (56Carr A. Biggin M.D. EMBO J. 1999; 18: 1598-1608Crossref PubMed Scopus (76) Google Scholar). Interaction of NRL with CRX in the two test systems (yeast two-hybrid and GST pull-down) did not require the presence of RPPR sequence elements. The addition of micrococcal nuclease to the GST pull-down reactions (in order to remove DNA template) did not significantly alter the interaction of CRX with NRL. This is different from the interaction of AP1 and NFAT, which occurs only when their cognate DNA binding region is present to form a quarternary complex (47Chen L. Glover J.N.M. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (414) Google Scholar, 59Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar). Our studies raise the possibility that the two activator proteins (NRL and CRX) form a stable complex by directly interacting with each other and probably with other proteins prior to their binding to their cognate cis-sequence elements in RPPR. CRX is shown to bind to Ret-4, BAT-1, and Ret1 elements in RPPR in vitro (31Chen S. Wang Q. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). We hypothesize that a stable NRL-CRX complex would influence the recognition of Ret-4 and BAT-1 flanking the NRE and provide binding sequence specificity during the organization of the rhodopsin enhanceosome. In addition, this interaction may result in cooperative and efficient DNA binding and explain their synergistic transactivation of the rhodopsin promoter. Mobility shift and DNase I experiments using the NRL and CRX proteins may help to confirm if the distribution of CRX on RPPR is influenced by NRL. The ability of NRL and CRX to physically interact with each other correlates well with their functional synergistic interaction at the rhodopsin promoter and illustrates one possible mechanism of context-dependent transcriptional regulation (60Fry C.J. Farnham P.J. J. Biol. Chem. 1999; 274: 29583-29586Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This interaction also points to cross-talk among different signal transduction pathways that activate or modulate rhodopsin expression in developing and mature photoreceptors. Further investigations are in progress to identify and characterize other activator proteins that are also involved in the formation of the rhodopsin enhanceosome. Elucidation of extracellular factors that influence the expression and activity of NRL, CRX, and these other factors should provide important new insights into the regulation of rhodopsin expression. We are grateful to Dr. Ching-Hwa Sung (Dyson Vision Research Institute, Cornell University Medical College, New York) for providing the bovine retina cDNA library in pACTII vector. We thank Drs. R. Daniel Gietz and Anna Evans for constructive advice and discussions and thank Sharyn Ferrara for assistance in preparing the manuscript.

Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further up-regulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

purpose. To assess the neuroprotective effect of virally mediated overexpression of ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in experimental rat glaucoma. methods. Laser-induced glaucoma was produced in one eye of 224 Wistar rats after injection of adenoassociated viral vectors (type 2) containing either CNTF, BDNF, or both, with saline-injected eyes and noninjected glaucomatous eyes serving as the control. IOP was measured with a hand-held tonometer, and semiautomated optic nerve axon counts were performed by masked observers. IOP exposure over time was adjusted in multivariate regression analysis to calculate the effect of CNTF and BDNF. results. By multivariate regression, CNTF had a significant protective effect, with 15% less RGC axon death (P < 0.01). Both combined CNTF-BDNF and BDNF overexpression alone had no statistically significant improvement in RGC axon survival. By Western blot, there was a quantitative increase in CNTF and BDNF expression in retinas exposed to single viral vectors carrying each gene, but no increase with sequential injection of both vectors. conclusions. These data confirm that CNTF can exert a protective effect in experimental glaucoma. The reason for the lack of observed effect in the BDNF overexpression groups is unclear, but it may be a function of the level of neurotrophin expression achieved.

To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli β-galactosidase gene (IacZ). Upstream sequences extending from −2174 to +70 bp, from −734 to +70 bp, and from −222 to +70 by direct photoreceptor-specific expression. All three −2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5′ of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.

Hypoxia inducible factor-1 (HIF-1) is a transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 transactivates multiple genes whose products play key roles in oxygen homeostasis, including vascular endothelial growth factor (VEGF). This study was designed to determine whether HIF-1 levels are increased in ischemic retina and whether there is a correlation with increased expression of VEGF.C57BL/6J mice were killed at time points that span retinal vascular development (PO to adult), or on postnatal day (P) 7 they were placed in a 75% oxygen environment for 5 days and then removed to room air and killed after 0, 2, or 6, or 24 hours and 5 or 14 days. Eyes were frozen, and retinas were isolated and used for immunoblot analysis, or eyes were sectioned for immunohisto chemical staining for HIF-1alpha or HIF-1beta, or for in situ hybridization for VEGF.Immunoblots of retinal lysates showed low levels of HIF-1alpha at PO that were markedly increased at P4, remained high throughout the period of retinal vascular development and then decreased to an intermediate level in adults. HIF-1beta levels were relatively constant at all time points. In mice with oxygen-induced ischemic retinopathy, HIF-1alpha levels were increased in the retina. The peak of increase occurred at 2 hours, and levels returned to baseline by 24 hours. Immunohistochemistry showed increased staining for HIF-1alpha throughout the hypoxic inner retina, but not in the normoxic outer retina. There was no modulation of HIF-1beta levels. There was constitutive expression of VEGF mRNA in the inner nuclear layer that was increased 6 hours after the onset of hypoxia and remained elevated for several days.There are increased levels of HIF-1alpha in ischemic retina that show temporal and spatial correlation with increased expression of VEGF. These findings are consistent with the hypothesis that HIF-1 plays a role in upregulation of VEGF in ischemic retina.

To use a rat model of optic nerve injury to differentiate primary and secondary retinal ganglion cell (RGC) injury.Under general anesthesia, a modified diamond knife was used to transect the superior one third of the orbital optic nerve in albino Wistar rats. The number of surviving RGC was quantified by counting both the number of cells retrogradely filled with fluorescent gold dye injected into the superior colliculus 1 week before nerve injury and the number of axons in optic nerve cross sections. RGCs were counted in 56 rats, with 24 regions examined in each retinal wholemount. Rats were studied at 4 days, 8 days, 4 weeks, and 9 weeks after transection. The interocular difference in RGCs was also compared in five control rats that underwent no surgery and in five rats who underwent a unilateral sham operation. It was confirmed histologically that only the upper optic nerve had been directly injured.At 4 and 8 days after injury, superior RGCs showed a mean difference from their fellow eyes of -30.3% and -62.8%, respectively (P = 0.02 and 0.001, t-test, n = 8 rats/group), whereas sham-operation eyes had no significant loss (mean difference between eyes = 1.7%, P = 0.74, t-test). At 8 days, inferior RGCs were unchanged from control, fellow eyes (mean interocular difference = -4.8%, P = 0.16, t-test). Nine weeks after transection, inferior RGC had 34.5% fewer RGCs than their fellow eyes, compared with 41.2% fewer RGCs in the superior zones of the injured eyes compared with fellow eyes. Detailed, serial section studies of the topography of RGC axons in the optic nerve showed an orderly arrangement of fibers that were segregated in relation to the position of their cell bodies in the retina.A model of partial optic nerve transection in rats showed rapid loss of directly injured RGCs in the superior retina and delayed, but significant secondary loss of RGCs in the inferior retina, whose axons were not severed. The findings confirm similar results in monkey eyes and provide a rodent model in which pharmacologic interventions against secondary degeneration can be tested.

Retinal ganglion cell (RGC) death in glaucoma involves apoptosis. Activation of caspases and abnormal processing of amyloid precursor protein (APP) are important events in other chronic neurodegenerations, such as Alzheimer's disease (AD). The retinal expression and activation of caspases and the patterns of caspase-3-mediated APP processing in ocular hypertensive models of rat glaucoma were investigated.RGC death was produced in one eye by chronic exposure to increased intraocular pressure (IOP) or by optic nerve transection. Elevated IOP was produced by obstruction of aqueous humor outflow with laser coagulation or limbal hypertonic saline injection. Caspase activity and APP processing in the retina were examined by RNase protection assay (RPA), immunocytochemistry, immunoblot assay, and colorimetric assay.RPA revealed elevations of caspase-3 mRNA, as well as other apoptosis-related mRNAs. Immunocytochemistry showed caspase-3 activation in RGCs damaged by ocular hypertension. The generation of the caspase-3-mediated APP cleavage product (DeltaC-APP) was also increased in ocular hypertensive RGCs. Western immunoblot assay and colorimetry revealed significantly more activated caspase-3 in ocular hypertensive retinas than in control retinas. The activated form of caspase-8, an initiator caspase, and amyloid-beta, a product of APP proteolysis and a component of senile plaques in AD, were detected in RGCs by immunohistochemistry significantly more often in ocular hypertensive than in control retinas. The amounts of full-length APP were reduced and amyloid-beta-containing fragments were increased in ocular hypertensive retinas by Western immunoblot assay.Rat RGCs subjected to chronic ocular hypertension demonstrate caspase activation and abnormal processing of APP, which may contribute to the pathophysiology of glaucoma.

The microRNA -183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated “ miR-183C GT/GT ,” using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b -wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

The inflammatory response that accompanies central nervous system (CNS) injury can affect neurological outcome in both positive and negative ways. In the optic nerve, a CNS pathway that normally fails to regenerate when damaged, intraocular inflammation causes retinal ganglion cells (RGCs) to switch into an active growth state and extend lengthy axons down the nerve. The molecular basis of this phenomenon is uncertain. A prior study showed that oncomodulin (Ocm), a Ca(2+)-binding protein secreted by a macrophage cell line, is a potent axon-promoting factor for RGCs. However, it is not known whether Ocm contributes to the physiological effects of intraocular inflammation in vivo, and there are conflicting reports in the literature regarding its expression and significance. We show here that intraocular inflammation causes infiltrative cells of the innate immune system to secrete high levels of Ocm, and that agents that prevent Ocm from binding to its receptor suppress axon regeneration. These results were verified in different strains, species, and experimental models, and establish Ocm as a potent growth-promoting signal between the innate immune system and neurons in vivo.

To determine the effect of pigment epithelium-derived factor (PEDF) in a mouse model of ischemia-induced retinal neovascularization and on vascular endothelial growth factor (VEGF)--induced migration and growth of cultured microvascular endothelial cells.Human recombinant PEDF was expressed in the human embryonic kidney 293 cell line and purified by ammonium sulfate precipitation and cation exchange chromatography. C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12 and then returned to room air. Mice received intravitreal injections of 2 microg PEDF in one eye and vehicle in the contralateral eye on P12 and P14. At P17, mice were killed and eyes enucleated for quantitation of retinal neovascularization. The mitogenic and motogeneic effects of VEGF on cultured bovine retinal and adrenal capillary endothelial cells were examined in the presence or absence of PEDF, using cell counts and migration assays.Two species of human recombinant PEDF, denoted A and B, were purified to apparent homogeneity. PEDF B appeared to comigrate on SDS-PAGE with PEDF from human vitreous samples. Changes in electrophoretic mobility after peptide-N-glycosidase F (PNGase F) digestion suggest that both PEDF forms contain N-linked carbohydrate. Analyses of the intact proteins by liquid chromatography-electrospray mass spectrometry (LC-ESMS) revealed the major molecular weight species for PEDF A (47,705 +/- 4) and B (46,757 +/- 5). LC-ESMS analysis of tryptic peptides indicated that PEDF A and B exhibit differences in glycopeptides containing N-acetylneuraminic acid (NeuAc) and N-acetylhexosamine (HexNAc). Intravitreal administration of either species of PEDF significantly inhibited retinal neovascularization (83% for PEDF A and 55% for PEDF B; P = 0.024 and 0.0026, respectively). PEDF A and B (20 nM) suppressed VEGF-induced retinal microvascular endothelial cell proliferation by 48.8% and 41.4%, respectively, after 5 days (P < 0.001) and VEGF-induced migration by 86.5% +/- 16.7% and 78.1% +/- 22.3%, respectively, after 4 hours (P = 0.004 and P = 0.008, respectively).These data indicate that elevated concentrations of PEDF inhibit VEGF-induced retinal endothelial cell growth and migration and retinal neovascularization. These findings suggest that localized administration of PEDF may be an effective approach for the treatment of ischemia-induced retinal neovascular disorders.

cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

The authors studied retinal gene expression changes in rats after experimental intraocular pressure elevation and optic nerve transection to elucidate molecular mechanisms of retinal ganglion cell (RGC) death.Translimbal laser photocoagulation was used to induce unilateral IOP elevation in 41 albino Wistar rats. In 38 additional animals, unilateral transection of the optic nerve was performed. Retinas were harvested 1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after each treatment, and total RNA was isolated. Pooled RNA from each time point was analyzed with rat genome arrays. Array results were confirmed by real-time PCR, and localization studies were performed using in situ hybridization for select genes.Genes that were upregulated in glaucoma, but not after transection, included Cyclin D2, Stat1, Stat3, c-Fos, Junb, Anxa1, Anxa 3, and CCAAT/enhancer binding protein (Cebp-delta). In glaucoma and transection models, the upregulation of c-Jun, Activating transcription factor 3, Heat shock protein 27, and Timp1 were observed. Comparisons among microarray databases were performed between our data and reports of retinal and optic nerve injury models in mice, rats, and monkeys.Gene expression changes specific to experimental glaucoma injury were identified. The present analysis supports the importance of neuroinflammation and the participation of the tumor necrosis factor alpha signaling pathway in glaucoma injury. The alterations observed include processes that are both protective of and detrimental to the survival of RGCs.

Transgenic mice with vascular endothelial growth factor (VEGF) driven by the rhodopsin promoter (rho/VEGF mice) develop neovascularization that originates from the deep capillary bed of the retina and grows into the subretinal space. In rho/VEGF mice, VEGF expression in photoreceptors begins between postnatal days 5 and 7, the period when the deep capillary bed is developing. An important question is whether or not the developmental stage of the deep capillary bed is critical for occurrence of neovascularization. Also, although rho/VEGF mice are extremely useful for the study of ocular neovascularization, there are some applications for which the early onset of VEGF expression is a disadvantage. In this study, we used the reverse tetracycline transactivator (rtTA) inducible promoter system coupled to either the rhodopsin or interphotoreceptor retinoid-binding protein (IRBP) promoter to control the time of onset of <i>VEGF</i> transgene expression in photoreceptors. In the absence of doxycycline, adult double-transgenic rho/rtTA-TRE/VEGF or IRBP/rtTA-TRE/VEGF mice showed little <i>VEGF</i> transgene expression and no phenotype. The addition of doxycycline to the drinking water resulted in prominent transgene expression and evidence of neovascularization within 3 to 4 days. Like rho/VEGF mice, the neovascularization originated from the deep capillary bed of the retina, but it was more extensive and caused outer retinal folds followed by total retinal detachment. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that the mice with inducible expression of VEGF that developed retinal detachment had much higher ocular levels of VEGF mRNA and protein compared to rho/VEGF mice that manifest a much milder phenotype. These data demonstrate that regardless of developmental stage of the vascular bed, increased expression of VEGF in the retina is sufficient to cause neovascularization, and high levels of expression cause severe neovascularization and traction retinal detachment. Mice with inducible expression of VEGF in the retina provide a valuable new model of ocular neovascularization.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

DNA methylation plays an important role in regulating gene expression during many biological processes. However, the mechanism of DNA-methylation-dependent gene regulation is not fully understood. Here, we explore two possible DNA methylation regulatory mechanisms with opposite modes of gene expression regulation.By comparing the genome-wide methylation and expression patterns in different tissues, we find that majority of tissue-specific differentially methylated regions (T-DMRs) are negatively correlated with expression of their associated genes (negative T-DMRs), consistent with the classical dogma that DNA methylation suppresses gene expression; however, a significant portion of T-DMRs are positively correlated with gene expression (positive T-DMRs). We observe that the positive T-DMRs have similar genomic location as negative T-DMRs, except that the positive T-DMRs are more enriched in the promoter regions. Both positive and negative T-DMRs are enriched in DNase I hypersensitivity sites (DHSs), suggesting that both are likely to be functional. The CpG sites of both positive and negative T-DMRs are also more evolutionarily conserved than the genomic background. Interestingly, the putative target genes of the positive T-DMR are enriched for negative regulators such as transcriptional repressors, suggesting a novel mode of indirect DNA methylation inhibition of expression through transcriptional repressors. Likewise, two distinct sets of DNA sequence motifs exist for positive and negative T-DMRs, suggesting that two distinct sets of transcription factors (TFs) are involved in positive and negative regulation mediated by DNA methylation.We find both negative and positive association between T-DMRs and gene expression, which implies the existence of two different mechanisms of DNA methylation-dependent gene regulation.

Abstract Age-related macular degeneration (AMD) is a significant cause of vision loss in the elderly. The extent to which epigenetic changes regulate AMD progression is unclear. Here we globally profile chromatin accessibility using ATAC-Seq in the retina and retinal pigmented epithelium (RPE) from AMD and control patients. Global decreases in chromatin accessibility occur in the RPE with early AMD, and in the retina of advanced disease, suggesting that dysfunction in the RPE drives disease onset. Footprints of photoreceptor and RPE-specific transcription factors are enriched in differentially accessible regions (DARs). Genes associated with DARs show altered expression in AMD. Cigarette smoke treatment of RPE cells recapitulates chromatin accessibility changes seen in AMD, providing an epigenetic link between a known risk factor for AMD and AMD pathology. Finally, overexpression of HDAC11 is partially responsible for the observed reduction in chromatin accessibility, suggesting that HDAC11 may be a potential new therapeutic target for AMD.

Abstract Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies. Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies. Disorders of photoreceptor degeneration contribute a significant burden to human genetic disease and can manifest in syndromic and nonsyndromic forms under a variety of inheritance models.1Inglehearn C.F. Molecular genetics of human retinal dystrophies.Eye (Lond). 1998; 12: 571-579Crossref PubMed Scopus (81) Google Scholar, 2Hims M.M. Diager S.P. Inglehearn C.F. Retinitis pigmentosa: Genes, proteins and prospects.Dev. Ophthalmol. 2003; 37: 109-125Crossref PubMed Scopus (93) Google Scholar, 3Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2018) Google Scholar, 4Daiger S.P. Bowne S.J. Sullivan L.S. Perspective on genes and mutations causing retinitis pigmentosa.Arch. Ophthalmol. 2007; 125: 151-158Crossref PubMed Scopus (327) Google Scholar At present, mutations in excess of 150 genes have been causally linked with retinal degeneration, many of which (35) have been shown to be necessary and sufficient to cause isolated retinitis pigmentosa (RP [MIM 268000]). Despite this remarkable progress, however, some 40% of the genetic burden of RP remains elusive,3Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2018) Google Scholar highlighting both the poignant genetic heterogeneity of the disorder and the presence of a likely significant number of private mutations. Gene discovery in RP has also highlighted an important mechanism found in numerous other traits, where ubiquitously expressed genes of apparently broad functionality cause a spatiotemporally restricted phenotype by harboring disease-causing mutations in tissue-specific (alternatively spliced) isoforms.5Kirschner R. Rosenberg T. Schultz-Heienbrok R. Lenzner S. Feil S. Roepman R. Cremers F.P. Ropers H.H. Berger W. RPGR transcription studies in mouse and human tissues reveal a retina-specific isoform that is disrupted in a patient with X-linked retinitis pigmentosa.Hum. Mol. Genet. 1999; 8: 1571-1578Crossref PubMed Scopus (111) Google Scholar, 6Gupta S.K. Leonard B.C. Damji K.F. Bulman D.E. A frame shift mutation in a tissue-specific alternatively spliced exon of collagen 2A1 in Wagner's vitreoretinal degeneration.Am. J. Ophthalmol. 2002; 133: 203-210Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7McAlinden A. Majava M. Bishop P.N. Perveen R. Black G.C. Pierpont M.E. Ala-Kokko L. Männikkö M. Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome.Hum. Mutat. 2008; 29: 83-90Crossref PubMed Scopus (36) Google Scholar At the severe end of the phenotypic spectrum, the classical RP phenotype of bone spicule pigmentation in the fundus, initially in the midperiphery and, in advanced stages, in the macula and fovea,8Heckenlively J.R. Yoser S.L. Friedman L.H. Oversier J.J. Clinical findings and common symptoms in retinitis pigmentosa.Am. J. Ophthalmol. 1988; 105: 504-511Abstract Full Text PDF PubMed Scopus (126) Google Scholar is also found in a number of syndromic traits, most prominently in Usher syndrome (MIM 276900), Bardet-Biedl syndrome (BBS [MIM 209900]), and other related ciliopathies.9Badano J.L. Mitsuma N. Beales P.L. Katsanis N. The ciliopathies: An emerging class of human genetic disorders.Annu. Rev. Genomics Hum. Genet. 2006; 7: 125-148Crossref PubMed Scopus (831) Google Scholar Recent studies have suggested that some genes that can cause syndromic retinal degeneration can also contribute to the development of isolated disease. For example, an intronic mutation that creates a splice-donor site and inserts a cryptic exon in NPHP6 (MIM 610142), the gene mutated in several ciliopathies including Joubert syndrome (JBTS5 [MIM 610188]), BBS, and Meckel syndrome (MKS4 [MIM 611134]),10Sayer J.A. Otto E.A. O'Toole J.F. Nurnberg G. Kennedy M.A. Becker C. Hennies H.C. Helou J. Attanasio M. Fausett B.V. et al.The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4.Nat. Genet. 2006; 38: 674-681Crossref PubMed Scopus (395) Google Scholar, 11Leitch C.C. Zaghloul N.A. Davis E.E. Stoetzel C. Diaz-Font A. Rix S. Alfadhel M. Al-Fadhel M. Lewis R.A. Eyaid W. et al.Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome.Nat. Genet. 2008; 40: 443-448Crossref PubMed Scopus (292) Google Scholar, 12Baala L. Audollent S. Martinovic J. Ozilou C. Babron M.C. Sivanandamoorthy S. Saunier S. Salomon R. Gonzales M. Rattenberry E. et al.Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome.Am. J. Hum. Genet. 2007; 81: 170-179Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar is sufficient to cause Leber congenital amaurosis (LCA10 [MIM 611755]), although the mechanism of phenotypic specificity is unclear.13den Hollander A.I. Koenekoop R.K. Yzer S. Lopez I. Arends M.L. Voesenek K.E. Zonneveld M.N. Strom T.M. Meitinger T. Brunner H.G. et al.Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis.Am. J. Hum. Genet. 2006; 79: 556-561Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar Here we report a splice-site mutation in a previously unknown exon of BBS8 (MIM 608132), one of the 14 genes that can cause BBS,14Zaghloul N.A. Katsanis N. Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy.J. Clin. Invest. 2009; 119: 428-437Crossref PubMed Scopus (269) Google Scholar that appears to be necessary and sufficient to cause nonsyndromic recessive RP in a consanguineous Pakistani family. In contrast to most known BBS8 mutations, which are predicted to significantly impact message stability and/or protein function,15Ansley S.J. Badano J.L. Blacque O.E. Hill J. Hoskins B.E. Leitch C.C. Kim J.C. Ross A.J. Eichers E.R. Teslovich T.M. et al.Basal body dysfunction is a likely cause of pleiotropic Bardet-Biedl syndrome.Nature. 2003; 425: 628-633Crossref PubMed Scopus (501) Google Scholar this mutation results in the skipping of 30 bp of an alternatively spliced exon (henceforth termed exon 2a), generating a shorter transcript that lacks 10 amino acids. Tissue-specific real-time PCR amplification suggests that exon 2a is expressed exclusively in the retina, whereas further studies of retinal tissue subsequent to laser capture microdissection indicate that this exon is expressed primarily in photoreceptors. These findings provide a potential mechanistic explanation for the restricted phenotype in the autosomal recessive RP (arRP) family and suggest that the role of this 10 amino acid sequence is critical for the retinal function of BBS8. As part of a larger study to identify new disease-causing genes for inherited visual diseases, we recruited a large consanguineous Pakistani family PKRP179 with four affected and seven unaffected siblings (Figure 1A ). Approval was obtained from the Institutional Review Board, and all participating subjects gave informed written consent consistent with the tenets of the Declaration of Helsinki. PKRP179 is from the southern region of the Punjab province of Pakistan, and detailed medical history was obtained by interviewing all family members. Patients were interviewed regarding age of first diagnosis, initial symptoms, progression, and any other ocular or systemic abnormalities. Ophthalmological examination with indirect dilated funduscopy was performed at Layton Rahmatullah Benevolent Trust Hospital, Lahore, Pakistan. Fundus photographs were acquired with a camera manufactured by Fuji. Electroretinography (ERG) measurements were recorded with equipment manufactured by LKC according to the standards of the International Society for Clinical Electrophysiology. Scotopic responses were recorded under dark-adapted conditions with a single bright flash stimulus, whereas the photopic responses were recorded under light-adapted conditions with a 30 Hz flicker stimulus. All affected individuals examined fulfilled the diagnostic criteria for diagnosis of RP, whereas retrospective analysis of available medical records was suggestive of early-onset RP in all four affected individuals (ages 2–4 at first diagnosis). We could detect no extraocular anomalies that typify syndromic RP. Fundus photographs of affected individuals showed typical changes of RP, including attenuation of retinal arteries and bone spicule pigment deposits in the midperiphery of the retina (Figure 2A ). This diagnosis was further confirmed by ERG; all four affected individuals, but none of the unaffected siblings or the unaffected mother, had typical RP changes on ERG, including loss of both rod and cone responses (representative data in Figure 2B). The size of the pedigree suggested sufficient power to achieve genome-wide statistical significance under a fully penetrant autosomal recessive mode of RP transmission (two-point LODmax = 3.44). We therefore conducted a genome-wide scan with 382 polymorphic short tandem repeat (STR) markers spaced equally across the genome. We performed two-point linkage analyses with the FASTLINK version of MLINK from the LINKAGE Program Package16Lathrop G.M. Lalouel J.M. Easy calculations of lod scores and genetic risks on small computers.Am. J. Hum. Genet. 1984; 36: 460-465PubMed Google Scholar and calculated maximum LOD scores with ILINK. Autosomal recessive RP was analyzed as a fully penetrant trait with a disease allele frequency of 0.001. The marker order and distances between the markers were obtained from the Marshfield database and the National Center for Biotechnology Information chromosome 14 sequence maps. Equal allele frequencies were assumed for the initial genome scan, whereas for fine mapping, allele frequencies were derived from 96 unrelated and unaffected individuals. We identified significant linkage with a single marker, D14S256, which yielded a two-point LOD score of 3.12 at θ = 0 (Figure 1B; Table 1). Additional STRs were selected to confirm this finding and to refine the critical interval; we observed two-point LOD scores of 3.05, 3.09, 3.03, and 3.32 at θ = 0 with D14S610, D14S67, D14S68, and D14S1058, respectively.Table 1Two-Point LOD Scores of Family PKRP179MarkercMMb0.000.010.050.100.200.30ZmaxθmaxD14S61293.7687.27−∞−2.57−1.15−0.67−0.59−0.170.080.40D14S61095.8988.203.052.972.702.361.781.093.050.00D14S6795.8988.383.092.992.742.381.791.143.090.00D14S6895.8988.623.032.962.712.381.711.043.030.00D14S105895.8988.643.323.253.062.681.901.303.320.00D14S256∗STR markers included in the genome-wide scan.96.4289.213.123.062.812.491.831.143.120.00D14S1050∗STR markers included in the genome-wide scan.107.1392.91−∞−2.37−1.04−0.61−0.54−0.160.100.40∗ STR markers included in the genome-wide scan. Open table in a new tab Reconstruction of the haplotypes supports the linkage analysis, localizing the pathogenic locus to chromosome 14q (Figure 1), an area not associated previously with mapped or cloned isolated RP loci. We found recombination events in affected individual V:1 at D14S612, defining the proximal boundary. Similarly, there was distal recombination in unaffected individual V:10 at D14S1050, placing the critical interval in a 13.4 cM (5.6 Mb) region between D14S612 and D14S1050. Notably, alleles for all STRs within the interval were homozygous in all affected individuals, supporting a homozygosity by descent (Figure 1); retrospective examination of genome-wide genotypes showed this region to be the sole genomic segment uniquely homozygous for all four patients, but not for any of their seven unaffected siblings, confirming that we have mapped another arRP locus, RP51. The critical interval harbors more than 100 annotated genes according to the USCS database. Subsequent to prioritizing genes on the basis of their known function and available expression data in the retina, we systematically started sequencing each candidate in one affected and one unaffected individual. Primer pairs for individual exons in the critical interval were designed with the Primer3 program (sequences and amplification conditions are available upon request). The PCR primers for each exon were used for bidirectional sequencing with BigDye Terminator Ready reaction mix, and sequencing was performed on an ABI PRISM 3100 automated sequencer (Applied Biosystems). Sequencing results were assembled with ABI PRISM sequencing analysis software version 3.7 and analyzed with SeqScape (Applied Biosystems). The third transcript in our list was BBS8/TTC8, rendered a strong candidate because of its causal involvement in BBS, one of the major hallmarks of which is RP. Analysis of the BBS8 transcript with the UCSC Genome Browser and the expressed sequence tag (EST) databases indicated the possibility that, in addition to the known, previously reported exons, this gene might also contain an additional 30 bp exon adjacent to its canonical second exon. We therefore included this putative expressed fragment in our sequence analysis of the entire transcript and found a homozygous A to G substitution in intron 1: IVS1-2A>G (Figure 3A ). The substitution maps to the splice acceptor site of the new exon, labeled exon 2a, and is predicted to result in skipping this exon, causing a 10 amino acid deletion of the protein (Figure 3B). Segregation of this change showed it to be transmitted with the disease phenotype; all affected individuals were homozygous, whereas unaffected individuals were either heterozygous or homozygous for the wild-type allele. Moreover, we did not find this change in 384 Pakistani control chromosomes or 384 chromosomes of northern European descent. Finally, alignment of the genomic sequence of this region with all available genomic sequences in the databases showed the splice acceptor site to be completely invariant in 33 species (Figure 3C), adding further evidence to the pathogenic potential of the mutation. These results prompted us to revisit the family for a thorough clinical examination. We found no evidence for syndromic disease (Table 2): none of the four affected individuals had renal problems; their body mass index was in the normal range; there were no signs of polydactyly (or scarring from a possible early excision of extra digits); and we found no evidence of developmental delay, appreciable cognitive impairment, or inappropriate social behavior. Finally, we observed no evidence of midline defects (high arched palates) or facies reminiscent of BBS. Although we were unable to examine for primary or secondary sexual characteristics, the absence of all other features of BBS strongly suggested that this family does not manifest the syndrome.Table 2Summary of the Phenotypic Finding of Affected Individuals in Family PKRP179: Individuals V:1, V:5, V:7, and V:10Individual IDV:1V:5V:7V:10RP (age at the time of first detection)4 yrs3 yrs2 yrs3 yrsDevelopmental delayNoNoNoNoTongue morphologyNormalNormalNormalNormalDental architectureNormalNormalNormalNormalLearning disabilityNoNoNoNoSpastic paraplegiaNoneNoneNoneNoneKidney defectsNoneNoneNoneNoneBehavior: speechNormalNormalNormalNormalBehavior: walkNormalNormalNormalNormalPolydactylyNoneNoneNoneNoneColobomasNoneNoneNoneNoneType II diabetesNoneNoneNoneNoneDeafnessNoneNoneNoneNoneWeight (lb)15811312590Height (in)75687061Body mass index19.717.217.917.0Note: The weight and height of the affected individuals V:1, V: 5, V:7, and V:10 were recorded at ages 29, 24, 21, and 16, respectively. Open table in a new tab Note: The weight and height of the affected individuals V:1, V: 5, V:7, and V:10 were recorded at ages 29, 24, 21, and 16, respectively. Our clinical and genetic findings raised the conundrum of a ubiquitously expressed gene giving rise to an isolated phenotype. One possible explanation is that the splice mutation is an in-frame event, and therefore a hypomorph. However, we and others have found a number of hypomorphic alleles in BBS8 that appear to be genetically sufficient to cause BBS. Therefore, although we could not formally exclude this possibility, we noticed that exon 2a was present in five ESTs present in the human EST database: notably, the originating tissue in all five clones was the eye. Because 26 of 30 bases (and both splice sites) of this exon are conserved in the mouse, we amplified both predicted Bbs8 splice isoforms from a panel of 12 mouse tissues known to express Bbs8 (eye, cortex, cerebellum, heart, liver, lung, kidney, testes, muscle, pancreas, stomach, and thymus). We found the shorter isoform (verified, upon sequencing, to lack exon 2a) in all tissues tested; by contrast, a longer isoform containing the predicted 30 bp exon could only be detected in the eye (data not shown). To verify these data, we sought to perform real-time quantitative RT-PCR (qRT-PCR). We amplified total RNA with the RNeasy micro kit (QIAGEN), and cDNAs were reverse transcribed with superscript III (Invitrogen) with random hexamers. PCR amplification was carried out with 10 ng of cDNA, and all reactions were performed in an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). Reactions consisted of a 3 min denaturation at 95°C followed by 45 cycles of denaturation for 10 s at 95°C and 30 s annealing at 60°C, as well as 30 s elongation at 72°C. Data were analyzed with Bio-Rad iQ5 Standard Edition V 2.1 program. The amount of each splice isoform was normalized against GAPDH (MIM 138400). By using this protocol on a subset of the original tissues tested, we were able to replicate these data (Figures 4A and 4B ). The RP phenotype is thought to be driven primarily by photoreceptor degeneration. We therefore interrogated the expression of the Bbs8 long message in different retinal layers by obtaining pertinent tissue through laser-capture microdissection. Briefly, adult C57BL/6 mice were euthanized, and eyes were enucleated. After removing the lens, the eye cups were incubated in calcium/magnesium-free Hanks' balanced salt solution at 37°C for 20 min to facilitate separation of the retina from the pigment epithelium. Eye cups were then immersed in 10%, 15%, and 25% sucrose for 30 min each and were snap frozen in OCT/25% sucrose. The eye cups were sectioned at 7 μm and mounted onto slides with charged PEN-foil membranes (Leica Microsystems). Sections were fixed in 70% ice-cold ethanol for 30 s, rinsed in RNase-free water, stained with hematoxylin for 10 s, and dehydrated in 70% ethanol and 100% ethanol for 1 min each. Retinal pigment epithelium (RPE), outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) samples were taken independently with an LMD6000 laser capture microdissection microscope (Leica Microsystems). Using the material obtained from this process, we isolated and purified mRNA from the retinal pigment epithelium, the outer nuclear layer, the inner nuclear layer, and the ganglion cell layer. qRT-PCR from each layer with probes detecting each of the two Bbs8 isoforms showed that, in contrast to the short Bbs8 mRNA species, which was detectable ubiquitously in the retina, the expression of the Bbs8 isoform affected by the patient mutation was almost exclusive to the photoreceptor layer, with a barely detectable message in the INL (Figures 4C and 4D). Taken together, these data suggest that the long BBS8 isoform is likely important to the function of this protein in the photoreceptors and suggest that loss of the ONL enriched, retina-specific exon can explain the isolated RP phenotype in humans. Here we have reported the mapping of RP51 with the aid of a large, consanguineous Pakistani kindred and demonstrate that a homozygous splice-site mutation affecting the conserved splice-acceptor site of an alternative exon in BBS8, exon 2a, is sufficient to explain the disorder at this locus. Although we cannot exclude the formal possibility that another lesion under the linkage peak can explain the phenotype, sequencing of most visible (on the genomic sequence) exons in the region did not reveal plausible candidate changes. Moreover, the mutant allele affects a base pair conserved completely in all 33 species in which this genomic segment is detectable, segregates with the disorder, and is absent from >1000 controls of various ethnicities (including 384 Pakistani control chromosomes), HapMap, and all available human genomes. Analysis of RP cohorts from Pakistan (n = 192), northern Europe (n = 302), and China (n = 192) did not identify additional lesions in this residue (or elsewhere in exon 2a), consistent with the suggestion that, with the exception of a few major loci, private mutations will constitute the majority of the missing 40% load of arRP.3Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2018) Google Scholar, 4Daiger S.P. Bowne S.J. Sullivan L.S. Perspective on genes and mutations causing retinitis pigmentosa.Arch. Ophthalmol. 2007; 125: 151-158Crossref PubMed Scopus (327) Google Scholar Detailed clinical examination of this family could exclude extraocular signs of BBS, indicating that, in contrast to all other BBS8 mutations, this allele only affects the function of the BBS8 protein in the photoreceptor. Although we cannot formally exclude the possibility that a separate cis or trans allele might convey protection against the nonretinal BBS phenotypes, that model is difficult to reconcile with the presence of isolated RP in all four affected siblings. Rather, our finding of expression of the affected exon exclusively in the retina, and almost uniquely in the ONL, suggests that the most likely explanation for our data is that failure of inclusion of exon 2a specifically impedes the function of a photoreceptor-specific (or heavily enriched) BBS8 protein isoform. The absence of this isoform from accessible patient tissues naturally precludes the direct study of the mutant message, and, in that context, it might be important to model this allele in the mouse as a means of both confirming our observations and developing a mechanistic model. Phenotypic restriction in broadly expressed genes driven by tissue-specific alternative splicing is not new. In the context of RP, a retina-specific exon of retinitis pigmentosa GTPase regulator (RPGR [MIM 312610]) harbors pathogenic mutations present in patients with isolated RP.5Kirschner R. Rosenberg T. Schultz-Heienbrok R. Lenzner S. Feil S. Roepman R. Cremers F.P. Ropers H.H. Berger W. RPGR transcription studies in mouse and human tissues reveal a retina-specific isoform that is disrupted in a patient with X-linked retinitis pigmentosa.Hum. Mol. Genet. 1999; 8: 1571-1578Crossref PubMed Scopus (111) Google Scholar Similarly, a frame-shifting mutation in an alternatively spliced exon of COL2A1 (MIM 120140) that is expressed specifically in vitreous collagen mRNA has been implicated in autosomal-dominant rhegmatogenous retinal detachment (MIM 609508), with the patients escaping the broader phenotypes associated with canonical COL2A1 mutations in Stickler syndrome (MIM 108300).6Gupta S.K. Leonard B.C. Damji K.F. Bulman D.E. A frame shift mutation in a tissue-specific alternatively spliced exon of collagen 2A1 in Wagner's vitreoretinal degeneration.Am. J. Ophthalmol. 2002; 133: 203-210Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7McAlinden A. Majava M. Bishop P.N. Perveen R. Black G.C. Pierpont M.E. Ala-Kokko L. Männikkö M. Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome.Hum. Mutat. 2008; 29: 83-90Crossref PubMed Scopus (36) Google Scholar Such examples add to the weight of evidence whereby variation of the mature mRNA message, through alternative splicing and editing, can specify organ and/or cell type pathology. Computational modeling of the additional 10 amino acid sequence with a variety of protein structure/domain programs did not detect any structural domain; as such, there are no immediate clues as to why the inclusion of exon 2a conveys photoreceptor-specific signaling properties to the protein. Nonetheless, we speculate that an understanding of the additional properties conferred to BBS8 by this extra sequence will inform the pathomechanism of retinal degeneration not only for some forms of arRP but also for BBS and other ciliopathies, which will lead to better therapeutics and treatments. We gratefully acknowledge the family members who donated samples to make this work possible. Financial support was provided by the Ministry of Science and Technology, Islamabad Pakistan, National Institutes of Health grants R01HD04260, R01DK072301, R01DK075972 (N.K.), and R01EY007142 (S.P.D.), the Macular Vision Research Foundation, and the Foundation Fighting Blindness (N.K.). N.K. is the George R. Brumley Professor. The URLs for data presented herein are as follows:International HapMap Project, http://www.hapmap.org/Marshfield Clinic Research Foundation, http://www.marshfieldclinic.org/research/pages/index.aspxNational Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim/Primer3 Input (version 0.4.0), http://frodo.wi.mit.edu/primer3Retinal Information Network (RetNet), http://www.retnet.orgSimple Modular Architecture Research Tool (SMART), http://smart.embl-heidelberg.de/UCSC Genome Browser, http://genome.ucsc.edu/

A common haplotype on 10q26 influences the risk of age-related macular degeneration (AMD) and encompasses two genes, LOC387715 and HTRA1. Recent data have suggested that loss of LOC387715, mediated by an insertion/deletion (in/del) that destabilizes its message, is causally related with the disorder. Here we show that loss of LOC387715 is insufficient to explain AMD susceptibility, since a nonsense mutation (R38X) in this gene that leads to loss of its message resides in a protective haplotype. At the same time, the common disease haplotype tagged by the in/del and rs11200638 has an effect on the transcriptional upregulation of the adjacent gene, HTRA1. These data implicate increased HTRA1 expression in the pathogenesis of AMD and highlight the importance of exploring multiple functional consequences of alleles in haplotypes that confer susceptibility to complex traits.

To study sequential changes in retinal ganglion cell (RGC) morphology in mice after optic nerve crush and after induction of experimental glaucoma.Nerve crush or experimental glaucoma was induced in mice that selectively express yellow fluorescent protein (YFP) in RGCs. Mice were euthanized 1, 4, and 9 days after crush and 1, 3, and 6 weeks after induction of glaucoma by bead injection. All YFP-RGCs were identified in retinal whole mounts. Then confocal images of randomly selected RGCs were quantified for somal fluorescence brightness, soma size, neurite outgrowth, and dendritic complexity (Sholl analysis).By 9 days after crush, 98% of RGC axons died and YFP-RGCs decreased by 64%. After 6 weeks of glaucoma, 31% of axons died, but there was no loss of YFP-RGC bodies. All crush retinas combined had significant decreases in neurite outgrowth parameters (P ≤ 0.036, generalized estimating equation [GEE] model) and dendritic complexity was lower than controls (P = 0.017, GEE model). There was no change in RGC soma area after crush. In combined glaucoma data, the RGC soma area was larger than control (P = 0.04, GEE model). At 3 weeks, glaucoma RGCs had significantly larger values for dendritic structure and complexity than controls (P = 0.044, GEE model), but no statistical difference was found at 6 weeks.After nerve crush, RGCs and axons died rapidly, and dendritic structure decreased moderately in remaining RGCs. Glaucoma caused an increase in RGC dendrite structure and soma size at 3 weeks.

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986.

The CAV1/CAV2 (caveolin 1 and caveolin 2) genomic region previously was associated with primary open-angle glaucoma (POAG), although replication among independent studies has been variable. The aim of this study was to assess the association between CAV1/CAV2 single nucleotide polymorphisms (SNPs) and POAG in a large case-control dataset and to explore associations by gender and pattern of visual field (VF) loss further.Case-control study.We analyzed 2 large POAG data sets: the Glaucoma Genes and Environment (GLAUGEN) study (976 cases, 1140 controls) and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium (2132 cases, 2290 controls).We studied the association between 70 SNPs located within the CAV1/CAV2 genomic region in the GLAUGEN and NEIGHBOR studies, both genotyped on the Illumina Human 660WQuadv1C BeadChip array and imputed with the Markov Chain Haplotyping algorithm using the HapMap 3 reference panel. We used logistic regression models of POAG in the overall population and separated by gender, as well as by POAG subtypes defined by type of VF defect (peripheral or paracentral). Results from GLAUGEN and NEIGHBOR were meta-analyzed, and a Bonferroni-corrected significance level of 7.7 × 10(-4) was used to account for multiple comparisons.Overall POAG, overall POAG by gender, and POAG subtypes defined by pattern of early VF loss.We found significant associations between 10 CAV1/CAV2 SNPs and POAG (top SNP, rs4236601; pooled P = 2.61 × 10(-7)). Of these, 9 were significant only in women (top SNP, rs4236601; pooled P = 1.59 × 10(-5)). Five of the 10 CAV1/CAV2 SNPs were associated with POAG with early paracentral VF (top SNP, rs17588172; pooled P = 1.07 × 10(-4)), and none of the 10 were associated with POAG with peripheral VF loss only or POAG among men.CAV1/CAV2 SNPs were associated significantly with POAG overall, particularly among women. Furthermore, we found an association between CAV1/CAV2 SNPs and POAG with paracentral VF defects. These data support a role for caveolin 1, caveolin 2, or both in POAG and suggest that the caveolins particularly may affect POAG pathogenesis in women and in patients with early paracentral VF defects.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and is influenced by genetic and environmental factors. Recent genome-wide association studies (GWAS) reported associations with IOP at TMCO1 and GAS7, and with primary open-angle glaucoma (POAG) at CDKN2B-AS1, CAV1/CAV2, and SIX1/SIX6. To identify novel genetic variants and replicate the published findings, we performed GWAS and meta-analysis of IOP in >6,000 subjects of European ancestry collected in three datasets: the NEI Glaucoma Human genetics collaBORation, GLAUcoma Genes and ENvironment study, and a subset of the Age-related Macular Degeneration-Michigan, Mayo, AREDS and Pennsylvania study. While no signal achieved genome-wide significance in individual datasets, a meta-analysis identified significant associations with IOP at TMCO1 (rs7518099-G, p = 8.0 × 10−8). Focused analyses of five loci previously reported for IOP and/or POAG, i.e., TMCO1, CDKN2B-AS1, GAS7, CAV1/CAV2, and SIX1/SIX6, revealed associations with IOP that were largely consistent across our three datasets, and replicated the previously reported associations in both effect size and direction. These results confirm the involvement of common variants in multiple genomic regions in regulating IOP and/or glaucoma risk.

Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.

Age-related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell-based therapies. Because of their indefinite self-renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE-like cells. However, most protocols developed to date for deriving RPE cells from hPSCs involve time- and labor-consuming manual steps, which hinder their use in biomedical applications requiring large amounts of differentiated cells. Here, we describe a simple and scalable protocol for the generation of RPE cells from hPSCs that is less labor-intensive. After amplification by clonal propagation using a myosin inhibitor, differentiation was induced in monolayers of hPSCs, and the resulting RPE cells were purified by two rounds of whole-dish single-cell passage. This approach yields highly pure populations of functional hPSC-derived RPE cells that display many characteristics of native RPE cells, including proper pigmentation and morphology, cell type-specific marker expression, polarized membrane and vascular endothelial growth factor secretion, and phagocytic activity. This work represents a step toward mass production of RPE cells from hPSCs.

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions.

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

Abstract Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

Abstract Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterized by varying degrees of secondary neurodegeneration. Retinal ganglion cells (RGC) are lost in MS in association with optic neuritis but the mechanisms of neuronal injury remain unclear. Complement component C3 has been implicated in retinal and cerebral synaptic pathology that may precede neurodegeneration. Herein, we examined post-mortem MS retinas, and then used a mouse model, experimental autoimmune encephalomyelitis (EAE), to examine the role of C3 in the pathogenesis of RGC loss associated with optic neuritis. First, we show extensive C3 expression in astrocytes (C3+/GFAP+ cells) and significant RGC loss (RBPMS+ cells) in post-mortem retinas from people with MS compared to retinas from non-MS individuals. A patient with progressive MS with a remote history of optic neuritis showed marked reactive astrogliosis with C3 expression in the inner retina extending into deeper layers in the affected eye more than the unaffected eye. To study whether C3 mediates retinal degeneration, we utilized global C3–/– EAE mice and found that they had less RGC loss and partially preserved neurites in the retina compared with C3+/+ EAE mice. C3–/– EAE mice had fewer axonal swellings in the optic nerve, reflecting reduced axonal injury, but had no changes in demyelination or T cell infiltration into the CNS. Using a C3-tdTomato reporter mouse line, we show definitive evidence of C3 expression in astrocytes in the retina and optic nerves of EAE mice. Conditional deletion of C3 in astrocytes showed RGC protection replicating the effects seen in the global knockouts. These data implicate astrocyte C3 expression as a critical mediator of retinal neuronal pathology in EAE and MS, and are consistent with recent studies showing C3 gene variants are associated with faster rates of retinal neurodegeneration in human disease.

Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases.Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2',3'-cyclic nucleotide 3'-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4'6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.

Although mammalian retinal ganglion cells (RGCs) normally cannot regenerate axons nor survive after optic nerve injury, this failure is partially reversed by inducing sterile inflammation in the eye. Infiltrative myeloid cells express the axogenic protein oncomodulin (Ocm) but additional, as-yet-unidentified, factors are also required. We show here that infiltrative macrophages express stromal cell–derived factor 1 (SDF1, CXCL12), which plays a central role in this regard. Among many growth factors tested in culture, only SDF1 enhances Ocm activity, an effect mediated through intracellular cyclic AMP (cAMP) elevation and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation. SDF1 deficiency in myeloid cells (CXCL12flx/flxLysM-Cre−/+ mice) or deletion of the SDF1 receptor CXCR4 in RGCs (intraocular AAV2-Cre in CXCR4flx/flx mice) or SDF1 antagonist AMD3100 greatly suppresses inflammation-induced regeneration and decreases RGC survival to baseline levels. Conversely, SDF1 induces optic nerve regeneration and RGC survival, and, when combined with Ocm/cAMP, SDF1 increases axon regeneration to levels similar to those induced by intraocular inflammation. In contrast to deletion of phosphatase and tensin homolog (Pten), which promotes regeneration selectively from αRGCs, SDF1 promotes regeneration from non-αRGCs and enables the latter cells to respond robustly to Pten deletion; however, SDF1 surprisingly diminishes the response of αRGCs to Pten deletion. When combined with inflammation and Pten deletion, SDF1 enables many RGCs to regenerate axons the entire length of the optic nerve. Thus, SDF1 complements the effects of Ocm in mediating inflammation-induced regeneration and enables different RGC subtypes to respond to Pten deletion.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Okamoto, N; Tobe, T; Hackett, S F; Ozaki, H; Vinores, M A; La Rochelle, W; Zack, D J; Campochiaro, P A Author Information

Retinoids play a critical role in vision, as well as in development and cellular differentiation. β,β-Carotene-15,15′-dioxygenase (Bcdo), the enzyme that catalyzes the oxidative cleavage of β,β-carotene into two retinal molecules, plays an important role in retinoid synthesis. We report here the first cloning of a mammalian Bcdo. Human BCDO encodes a protein of 547 amino acid residues that demonstrates 68% identity with chicken Bcdo. It is expressed highly in the retinal pigment epithelium (RPE) and also in kidney, intestine, liver, brain, stomach, and testis. The gene spans approximately 20 kb, is composed of 11 exons and 10 introns, and maps to chromosome 16q21–q23. A mouse orthologue was also identified, and its predicted amino acid sequence is 83% identical with human BCDO. Biochemical analysis of baculovirus expressed human BCDO demonstrates the predicted β,β-carotene-15,15′-dioxygenase activity. The expression pattern of BCDO suggests that it may provide a local supplement to the retinoids available to photoreceptors, as well as a supplement to the retinoid pools utilized elsewhere in the body. In addition, the finding that many of the enzymes involved in retinoid metabolism are mutated in retinal degenerations suggests that BCDO may also be a candidate gene for retinal degenerative disease.

Intravitreal injection of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF2) promotes survival of photoreceptors exposed to various types of insults, but it is not known if these survival-promoting effects occur by direct action of the factors on photoreceptors or indirectly through the activation of other cells. In this study, the authors have sought to address this issue by determining which cells in the retina show evidence of activated intracellular signaling pathways acutely and at longer time points after intravitreal injection of these agents.Retinas were removed from C57BL/6J mice at 1, 6, or 24 hours after intravitreal injection of 1 microg of human BDNF, rat CNTF, human FGF2, or human transforming growth factor-alpha (TGFalpha), and immunohistochemically stained for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated cAMP responsive element binding protein (pCREB), or c-fos. Retinal organ cultures were incubated with 10 ng/ml of BDNF, CNTF, FGF2, or TGFalpha for 10 or 30 minutes or 1, 3, or 6 hours and then immunohistochemically stained for pERK, pCREB, or c-fos.Intravitreal injection of BDNF, CNTF, or FGF2 resulted in a rapid increase in pERK immunoreactivity in Müller cells and a rapid increase in c-fos immunoreactivity in Müller, amacrine, and ganglion cells. Immunoreactivity for pERK and c-fos returned to baseline in all retinal cells at 6 or 24 hours after injection, but there was increased staining for glial fibrillary acidic protein (GFAP) in Müller cells at these time points. At no time after injection was there any staining for pERK or c-fos in photoreceptors. Similarly, retinal explants treated with FGF2, BDNF, or CNTF showed increased staining for pCREB, pERK, and c-fos in cells of the inner retina, but not photoreceptors.These data support the hypothesis that BDNF, CNTF, and FGF2 exert their effects on photoreceptors by acting indirectly through activation of Müller cells and perhaps other nonphotoreceptor cells.

In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between −40 and −84 base pairs (bp) and another between −84 and −130 bp. Activity of the −40 to −84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The −84- to −130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression. In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between −40 and −84 base pairs (bp) and another between −84 and −130 bp. Activity of the −40 to −84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The −84- to −130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.

Tissue-specific gene expression is generally regulated by more than a single transcription factor (TF). Multiple TFs work in concert to achieve tissue specificity. In order to explore these complex TF interaction networks, we performed a large-scale analysis of TF interactions for 30 human tissues. We first identified tissue-specific genes for 30 tissues based on gene expression databases. We then evaluated the relationships between TFs using the relative position and co-occurrence of their binding sites in the promoters of tissue-specific genes. The predicted TF-TF interactions were validated by both known protein-protein interactions and co-expression of their target genes. We found that our predictions are enriched in known protein-protein interactions (>80 times that of random expectation). In addition, we found that the target genes show the highest co-expression in the tissue of interest. Our findings demonstrate that non-tissue specific TFs play a large role in regulation of tissue-specific genes. Furthermore, they show that individual TFs can contribute to tissue specificity in different tissues by interacting with distinct TF partners. Lastly, we identified several tissue-specific TF clusters that may play important roles in tissue-specific gene regulation.

There are no effective treatments for inherited retinal degenerations, which are prevalent causes of visual disability. Several proteins promote the survival of various types of neurons, and increasing expression of one or more of these survival factors is a promising strategy for a new treatment. Studies examining the effects of intravitreous injections of brain-derived neurotrophic factor (BDNF) in models of inherited retinal degenerations have suggested that BDNF has little survival-promoting activity for photoreceptors. In this study, we generated double transgenic mice with doxycycline-inducible expression of BDNF in the retina. In a model of primary rod photoreceptor degeneration, expression of BDNF resulted in significant delay in photoreceptor cell death and maintenance of retinal function assessed by electroretinogram recordings. Expression of BDNF also caused strong protection of photoreceptors from oxidative damage-induced cell death. These data suggest that continuous expression of BDNF, unlike intravitreous injections, results in morphologic and functional benefit in animal models of inherited retinal degeneration. Double transgenic mice with inducible expression of survival factors provide valuable tools for selection of survival factor candidates for gene therapy.

Adeno-associated viral (AAV) vectors have been used to express several different proteins in the eye. The purpose of this study was to determine whether AAV-mediated intraocular gene transfer of pigment epithelium-derived factor (PEDF) inhibits the development of choroidal neovascularization (CNV) in a murine model.C57BL/6 mice were given intravitreous or subretinal injections of a PEDF expression construct packaged in an AAV vector (AAV-chicken beta-actin promoter-exon 1-intron 1[CBA]-PEDF) or control vector (AAV-CBA-green fluorescent protein[GFP]). After 4 or 6 weeks, the Bruch's membrane was ruptured by laser photocoagulation at three sites in each eye. After 14 days, the area of CNV at each rupture site was measured by image analysis. Intraocular levels of PEDF were measured by enzyme-linked immunosorbent assay.Four to six weeks after intraocular injection of AAV-CBA-PEDF, levels of PEDF in whole-eye homogenates were 6 to 70 ng. The average area of CNV at sites of the Bruch's membrane rupture showed no significant difference in eyes injected with AAV-CBA-PEDF compared with uninjected eyes. In contrast, 4 to 6 weeks after intraocular injection of 1.5 x 10(9) or 2.0 x 10(10) particles of AAV-CBA-PEDF, the area of CNV at the Bruch's membrane rupture sites had significantly decreased compared with CNV area at rupture sites in eyes injected with AAV-CBA-GFP.These data suggest that intraocular expression of PEDF or other antiangiogenic proteins with AAV vectors may provide a new treatment approach for ocular neovascularization.

Purpose.: MicroRNAs (miRNAs) are short, noncoding transcripts that negatively regulate gene expression. They are implicated in diverse cellular processes. The purpose of this study was to obtain a global expression profile of miRNAs in the developing retina and identify differences in miRNA expression between adult rod and cone photoreceptors. Methods.: Locked nucleic acid (LNA) microarrays were used to investigate the miRNA transcriptome of the developing mouse retina and brain. Real-time PCR was used to validate the array findings. Laser capture microdissection was used to determine the miRNA spatial pattern of expression. Results.: One hundred thirty-eight miRNAs were expressed at at least one of the investigated time points. Several miRNAs showed significant changes in expression between embryonic day 15 and adult age in both retina and brain. Cluster analysis identified subgroups of miRNAs showing defined expression profiles. Globally, correlation of expression was higher, with increasing sequence similarity of the mature miRNAs. The miRNAs with identical seed sequences exhibited highly correlated expression profiles. The co-expression of selected host gene and intronic miRNA pairs was confirmed in adult retina. In some cases, expression profiles of miRNAs showed weak correlation with those of their host transcripts, suggesting posttranscriptional regulation of miRNAs during development. In addition, the miRNA transcriptome of rod- and cone-dominant retinas showed only minor differences, and no miRNAs specific for either cell-type were identified. Conclusions.: Global expression profiling revealed dozens of miRNAs with significant expression changes in the developing retina. Precise patterns of expression of miRNAs suggest their specific roles in development.

MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif. Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties. The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology.

The E46K is a point mutation in alpha-synuclein (alpha-syn) that causes familial Parkinsonism with Lewy body dementia. We have now generated a cell model of Parkinsonism/Parkinson's disease (PD) and demonstrated cell toxicity after expression of E46K in the differentiated PC12 cells. E46K alpha-syn inhibited proteasome activity and induced mitochondrial depolarization in the cell model. Baicalein has been reported to inhibit fibrillation of wild type alpha-syn in vitro, and to protect neurons against several chemical-induced models of PD. We now report that baicalein significantly attenuated E46K-induced mitochondrial depolarization and proteasome inhibition, and protected cells against E46K-induced toxicity in a cell model of PD. Baicalein also reduced E46K fibrilization in vitro, with a concentration-dependent decrease in beta sheet conformation, though it increased some oligomeric species, and decreased formation of E46K alpha-syn-induced aggregates and rescued toxicity in N2A cells. Taken together, these data indicate that mitochondrial dysfunction, proteasome inhibition and specific aspects of abnormal E46K aggregation accompany E46K alpha-syn-induced cell toxicity, and baicalein can protect as well as altering aggregation properties. Baicalein has potential as a tool to understand the relation between different aggregation species and toxicity, and might be a candidate compound for further validation by using in vivo alpha-syn genetic PD models.

To investigate whether the 2 subtypes of advanced age-related macular degeneration (AMD), choroidal neovascularization (CNV), and geographic atrophy (GA) segregate separately in families and to identify which genetic variants are associated with these 2 subtypes.Sibling correlation study and genome-wide association study (GWAS).For the sibling correlation study, 209 sibling pairs with advanced AMD were included. For the GWAS, 2594 participants with advanced AMD subtypes and 4134 controls were included. Replication cohorts included 5383 advanced AMD participants and 15 240 controls.Participants had the AMD grade assigned based on fundus photography, examination, or both. To determine heritability of advanced AMD subtypes, a sibling correlation study was performed. For the GWAS, genome-wide genotyping was conducted and 6 036 699 single nucleotide polymorphisms (SNPs) were imputed. Then, the SNPs were analyzed with a generalized linear model controlling for genotyping platform and genetic ancestry. The most significant associations were evaluated in independent cohorts.Concordance of advanced AMD subtypes in sibling pairs and associations between SNPs with GA and CNV advanced AMD subtypes.The difference between the observed and expected proportion of siblings concordant for the same subtype of advanced AMD was different to a statistically significant degree (P = 4.2 × 10(-5)), meaning that in siblings of probands with CNV or GA, the same advanced subtype is more likely to develop. In the analysis comparing participants with CNV to those with GA, a statistically significant association was observed at the ARMS2/HTRA1 locus (rs10490924; odds ratio [OR], 1.47; P = 4.3 × 10(-9)), which was confirmed in the replication samples (OR, 1.38; P = 7.4 × 10(-14) for combined discovery and replication analysis).Whether CNV versus GA develops in a patient with AMD is determined in part by genetic variation. In this large GWAS meta-analysis and replication analysis, the ARMS2/HTRA1 locus confers increased risk for both advanced AMD subtypes, but imparts greater risk for CNV than for GA. This locus explains a small proportion of the excess sibling correlation for advanced AMD subtype. Other loci were detected with suggestive associations that differ for advanced AMD subtypes and deserve follow-up in additional studies.

Purpose To assess the association between single nucleotide polymorphisms (SNPs) of the gene region containing cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and glaucoma features among primary open-angle glaucoma (POAG) patients. Design Retrospective observational case series. Methods We studied associations between 10 CDKN2B-AS1 SNPs and glaucoma features among 976 POAG cases from the Glaucoma Genes and Environment (GLAUGEN) study and 1971 cases from the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium. For each patient, we chose the feature from the eye with the higher value. We created cohort-specific multivariable models for glaucoma features and then meta-analyzed the results. Results For 9 of the 10 protective CDKN2B-AS1 SNPs with minor alleles associated with reduced disease risk (eg, the G allele at rs2157719), POAG patients carrying these minor alleles had smaller cup-to-disc ratio (0.05 units smaller per G allele at diagnosis; 95% CI: −0.08, −0.03; P = 6.23E-05) despite having higher intraocular pressure (IOP) (0.70 mm Hg higher per G allele at DNA collection; 95% CI: 0.40, 1.00; P = 5.45E-06). For the 1 adverse rs3217992 SNP with minor allele A associated with increased disease risk, POAG patients with A alleles had larger cup-to-disc ratio (0.05 units larger per A allele at diagnosis; 95% CI: 0.02, 0.07; P = 4.74E-04) despite having lower IOP (−0.57 mm Hg per A allele at DNA collection; 95% CI: −0.84, −0.29; P = 6.55E-05). Conclusion Alleles of CDKN2B-AS1 SNPs, which influence risk of developing POAG, also modulate optic nerve degeneration among POAG patients, underscoring the role of CDKN2B-AS1 in POAG. To assess the association between single nucleotide polymorphisms (SNPs) of the gene region containing cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and glaucoma features among primary open-angle glaucoma (POAG) patients. Retrospective observational case series. We studied associations between 10 CDKN2B-AS1 SNPs and glaucoma features among 976 POAG cases from the Glaucoma Genes and Environment (GLAUGEN) study and 1971 cases from the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium. For each patient, we chose the feature from the eye with the higher value. We created cohort-specific multivariable models for glaucoma features and then meta-analyzed the results. For 9 of the 10 protective CDKN2B-AS1 SNPs with minor alleles associated with reduced disease risk (eg, the G allele at rs2157719), POAG patients carrying these minor alleles had smaller cup-to-disc ratio (0.05 units smaller per G allele at diagnosis; 95% CI: −0.08, −0.03; P = 6.23E-05) despite having higher intraocular pressure (IOP) (0.70 mm Hg higher per G allele at DNA collection; 95% CI: 0.40, 1.00; P = 5.45E-06). For the 1 adverse rs3217992 SNP with minor allele A associated with increased disease risk, POAG patients with A alleles had larger cup-to-disc ratio (0.05 units larger per A allele at diagnosis; 95% CI: 0.02, 0.07; P = 4.74E-04) despite having lower IOP (−0.57 mm Hg per A allele at DNA collection; 95% CI: −0.84, −0.29; P = 6.55E-05). Alleles of CDKN2B-AS1 SNPs, which influence risk of developing POAG, also modulate optic nerve degeneration among POAG patients, underscoring the role of CDKN2B-AS1 in POAG.

Purpose.: To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes. Methods.: A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography. Results.: The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity. Conclusions.: This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non–cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

Alternative splicing is highly regulated in tissue-specific and development-specific patterns, and it has been estimated that 15% of disease-causing point mutations affect pre-mRNA splicing. In this review, we consider the cis-acting splice site and trans-acting splicing factor mutations that affect pre-mRNA splicing and contribute to retinal degeneration. Numerous splice site mutations have been identified in retinitis pigmentosa (RP) and various cone-rod dystrophies. Mutations in alternatively spliced retina-specific exons of the widely expressed RPGR and COL2A1 genes lead primarily to X-linked RP and ocular variants of Stickler syndrome, respectively. Furthermore, mutations in general pre-mRNA splicing factors, such as PRPF31, PRPF8, and PRPF3, predominantly cause autosomal dominant RP. These findings suggest an important role for pre-mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule-based therapies, has the potential to lead to new treatments for retinal degenerative diseases.

Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.

The repurposed CRISPR–Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species. Current CRISPR-mediated genome-editing methods are limited by the requirement for a specific +1 nucleotide when using the U6 promoter to drive guide RNA synthesis. Now, Ranganathan et al.report a modification of the CRISPR–Cas9 system that more than doubles the number of targetable CRISPR sites within the human genome.

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes' neuroprotective efficacy on retinal ganglion cells (RGCs).MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell-derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes.MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance.MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.

The animal model experimental autoimmune encephalomyelitis (EAE) has been used extensively in the past to test mechanisms that target peripheral immune cells for treatment of multiple sclerosis (MS). While there have been some notable successes in relapsing MS, the development of therapies for progressive multiple sclerosis (MS) has been hampered by lack of an appropriate animal model. Further, the mechanisms underlying CNS inflammation and neuronal injury remain incompletely elucidated. It is known that the MOG 35–55 EAE mouse model does not have insidious behavioral progression as occurs in people with MS, but there is significant neuronal and axonal injury in EAE, as a result of the inflammation. In the present study, we describe the time course of glial activation and retinal neurodegeneration in the EAE model, and highlight the utility of studying the anterior visual pathway for modeling mechanisms of neuronal injury that may recapitulate critical aspects of the pathology described in people with MS following optic neuritis and subclinical optic neuropathy. We show that A1 neurotoxic astrocytes are prevalent in optic nerve tissue and retina, and are associated with subsequent RGC loss in the most commonly used form of the EAE model induced by MOG 35–55 peptide in C57/B6 mice. We developed a semi-automatic method to quantify retinal ganglion cells (RGC) and show that RGCs remain intact at peak EAE (PID 16) but are significantly reduced in late EAE (PID 42). Postsynaptic proteins and neurites were also compromised in the retina of late EAE mice. The retinal pathology manifests weeks after the microglial and astrocyte activation, which were prominent in optic nerve tissues at PID 16. Microglia expressed iNOS and had increased gene expression of C1q, TNF-α, and IL-1α. Astrocytes expressed high levels of complement component 3 and other genes associated with A1 neurotoxic astrocytes. Our data suggest that EAE can be used to study the pathobiology of optic neuropathy and to examine the preclinical neuroprotective effects of drugs that target activation of neurotoxic A1 astrocytes.

Genome-wide association studies (GWAS) for late stage age-related macular degeneration (AMD) have identified 52 independent genetic variants with genome-wide significance at 34 genomic loci. Typically, such an approach rarely results in the identification of functional variants implicating a defined gene in the disease process. We now performed a transcriptome-wide association study (TWAS) allowing the prediction of effects of AMD-associated genetic variants on gene expression. The TWAS was based on the genotypes of 16,144 late-stage AMD cases and 17,832 healthy controls, and gene expression was imputed for 27 different human tissues which were obtained from 134 to 421 individuals. A linear regression model including each individuals imputed gene expression data and the respective AMD status identified 106 genes significantly associated to AMD variants in at least one tissue (Q-value < 0.001). Gene enrichment analysis highlighted rather systemic than tissue- or cell-specific processes. Remarkably, 31 of the 106 genes overlapped with significant GWAS signals of other complex traits and diseases, such as neurological or autoimmune conditions. Taken together, our study highlights the fact that expression of genes associated with AMD is not restricted to retinal tissue as could be expected for an eye disease of the posterior pole, but instead is rather ubiquitous suggesting processes underlying AMD pathology to be of systemic nature.

Recent advances in stem cell engineering have led to an explosion in the use of organoids as model systems for studies in multiple biological disciplines. Together with breakthroughs in genome engineering and the various omics, organoid technology is making possible studies of human biology that were not previously feasible. For vision science, retinal organoids derived from human stem cells allow differentiating and mature human retinal cells to be studied in unprecedented detail. In this review, we examine the technologies employed to generate retinal organoids and how organoids are revolutionizing the fields of developmental and cellular biology as they pertain to the retina. Furthermore, we explore retinal organoids from a clinical standpoint, offering a new platform with which to study retinal diseases and degeneration, test prospective drugs and therapeutic strategies, and promote personalized medicine. Finally, we discuss the range of possibilities that organoids may bring to future retinal research and consider their ethical implications.

Retinal ganglion cell (RGC) replacement holds potential for restoring vision lost to optic neuropathy. Transplanted RGCs must undergo neuroretinal integration to receive afferent visual signals for processing and efferent transmission. To date, retinal integration following RGC transplantation has been limited. We sought to overcome key barriers to transplanted human stem cell-derived RGC integration. Following co-culture ex vivo on organotypic mouse retinal explants, human RGCs cluster and extend bundled neurites that remain superficial to the neuroretina, hindering afferent synaptogenesis. To enhance integration, we increased the cellular permeability of the internal limiting membrane (ILM). Extracellular matrix digestion using proteolytic enzymes achieved ILM disruption while minimizing retinal toxicity and preserving glial reactivity. ILM disruption is associated with dispersion rather than clustering of co-cultured RGC bodies and neurites, and increased parenchymal neurite ingrowth. The ILM represents a significant obstacle to transplanted RGC connectivity and its circumvention may be necessary for functional RGC replacement.

Advanced age-related macular degeneration (AMD) is a leading cause of blindness in Western countries. Causal, modifiable risk factors need to be identified to develop preventive measures for advanced AMD.To assess whether smoking, alcohol consumption, blood pressure, body mass index, and glycemic traits are associated with increased risk of advanced AMD.This study used 2-sample mendelian randomization. Genetic instruments composed of variants associated with risk factors at genome-wide significance (P < 5 × 10-8) were obtained from published genome-wide association studies. Summary-level statistics for these instruments were obtained for advanced AMD from the International AMD Genomics Consortium 2016 data set, which consisted of 16 144 individuals with AMD and 17 832 control individuals. Data were analyzed from July 2020 to September 2021.Smoking initiation, smoking cessation, lifetime smoking, age at smoking initiation, alcoholic drinks per week, body mass index, systolic and diastolic blood pressure, type 2 diabetes, glycated hemoglobin, fasting glucose, and fasting insulin.Advanced AMD and its subtypes, geographic atrophy (GA), and neovascular AMD.A 1-SD increase in logodds of genetically predicted smoking initiation was associated with higher risk of advanced AMD (odds ratio [OR], 1.26; 95% CI, 1.13-1.40; P < .001), while a 1-SD increase in logodds of genetically predicted smoking cessation (former vs current smoking) was associated with lower risk of advanced AMD (OR, 0.66; 95% CI, 0.50-0.87; P = .003). Genetically predicted increased lifetime smoking was associated with increased risk of advanced AMD (OR per 1-SD increase in lifetime smoking behavior, 1.32; 95% CI, 1.09-1.59; P = .004). Genetically predicted alcohol consumption was associated with higher risk of GA (OR per 1-SD increase of log-transformed alcoholic drinks per week, 2.70; 95% CI, 1.48-4.94; P = .001). There was insufficient evidence to suggest that genetically predicted blood pressure, body mass index, and glycemic traits were associated with advanced AMD.This study provides genetic evidence that increased alcohol intake may be a causal risk factor for GA. As there are currently no known treatments for GA, this finding has important public health implications. These results also support previous observational studies associating smoking behavior with risk of advanced AMD, thus reinforcing existing public health messages regarding the risk of blindness associated with smoking.

The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N -acetyltransferase (NAT) and hydroxyindole- O -methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene ( Atp7b ) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5′ regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE.

Mutations in the gene for guanylate cyclase–activating protein-1 (GCAP1) (<i>GUCA1A</i>) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of <i>GUCA1A</i>. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca²⁺ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca²⁺. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca²⁺ concentrations but fails to inactivate at high Ca²⁺ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca²⁺ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

Apoptosis is a form of genetically programmed cell death that can be induced by a variety of different stimuli. It is often referred to as a form of cellular suicide. Typically, apoptosis is characterized by the condensation and shrinkage of the cellular nucleus and cytoplasm, followed by the complete fragmentation of the cell and subsequent phagocytosis of the debris by surrounding cells. Although important during development, and also for maintaining homeostasis in some adult tissues, apoptosis can also be associated with disease processes. Recent laboratory studies indicate that apoptosis is a mechanism of cell death in several important ocular diseases including glaucoma, retinitis pigmentosa, cataract formation, retinoblastoma, retinal ischemia, and diabetic retinopathy. This review summarizes the results of these studies and provides a brief description of some of the key molecules that are involved in the genetic regulation of apoptosis. It is possible that a complete understanding of how these molecules function may someday lead to new treatment options aimed at blocking the death of cells in a variety of ocular diseases.

The Wnt/frizzled cell signaling pathway has been implicated in the determination of polarity in a number of systems, including the Drosophila retina. The vertebrate retina develops from an undifferentiated neuroepithelium into an organized and laminated structure that demonstrates a high degree of polarity at both the tissue and cellular levels. In the process of searching for molecules that are preferentially expressed by the vertebrate retinal pigment epithelium (RPE), we identified secreted frizzled-related protein 5 (SFRP5), a member of the SFRP family that appears to act by modulating Wnt signal transduction. SFRP5 is highly expressed by RPE cells, and is also expressed in the pancreas. Within the retina, the related molecule SFRP2 is expressed specifically by cells of the inner nuclear layer. Thus, photoreceptors are likely to be bathed by two opposing gradients of SFRP molecules. Consistent with SFRP5 's postulated role in modulating Wnt signaling in the retina, it inhibits the ability of Xwnt-8 mRNA to induce axis duplication in Xenopus embryos. The human SFRP5 gene consists of three coding exons and it maps to chromosome 10q24.1; human SFRP2 maps to 4q31.3. Based on the biology and complementary expression patterns of SFRP2 and SFRP5, we suggest that they may be involved in determining the polarity of photoreceptor, and perhaps other, cells in the retina.

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.