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1.  Cone-like Morphological, Molecular, and Electrophysiological Features of the Photoreceptors of the Nrl Knockout Mouse 
Purpose
To test the hypothesis that Nrl−/− photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysio-logical criteria.
Methods
The photoreceptor layer of fixed retinal tissue of 4-to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG.
Results
Outer segments of Nrl−/− photoreceptors (~7 μm) are shorter than those of wild-type (WT) rods (~25 μm) and cones (~15 μm); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin “clumping” in their nuclei, and mitochondria two times shorter than rods. Nrl−/− photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl−/− a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the λmax of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was ~50 ms, more than two times faster than that of rods.
Conclusions
Many morphological, molecular, and electrophys iological features of the Nrl−/− photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.
doi:10.1167/iovs.04-1427
PMCID: PMC4505833  PMID: 15914637
2.  Nrl-Knockout Mice Deficient in Rpe65 Fail to Synthesize 11-cis Retinal and Cone Outer Segments 
Purpose
To define rod and cone function further in terms of visual cycle mechanism, the retinal phenotype resulting from Rpe65 (retinoid isomerase I) deficiency in Nrl−/− mice having a single class of photoreceptors resembling wild-type cones was characterized and outcomes of retinoid supplementation evaluated.
Methods
Rpe65−/−/Nrl−/− mice were generated by breeding Rpe65−/− and Nrl−/− strains. Retinal histology, protein expression, retinoid content, and electroretinographic (ERG) responses were evaluated before and after treatment with 11-cis retinal by intraperitoneal injection.
Results
Retinas of young Rpe65−/−/Nrl−/− mice exhibited normal lamination, but lacked intact photoreceptor outer segments at all ages examined. Rpe65, Nrl, and rhodopsin were not detected, and S-opsin and M/L-opsin levels were reduced. Retinyl esters were the only retinoids present. In contrast, Nrl−/− mice exhibited decreased levels of retinaldehydes and retinyl esters, and elevated levels of retinols. ERG responses were elicited from Rpe65−/−/Nrl−/− mice only at the two highest intensities over a 4-log-unit range. Significant retinal thinning and outer nuclear layer loss occurred in Rpe65−/−/Nrl−/− mice with aging. Administration of exogenous 11-cis retinal did not rescue retinal morphology or markedly improve ERG responses.
Conclusions
The findings provide clarification of reported cone loss of function in Rpe65−/−/Nrl−/− mice, now showing that chromophore absence results in destabilized cone outer segments and rapid retinal degeneration. The data support the view that rod-dominant retinas do not have a cone-specific mechanism for 11-cis retinal synthesis and have potential significance for therapeutic strategies for rescue of cone-rich retinal regions affected by disease in the aging human population.
doi:10.1167/iovs.07-1234
PMCID: PMC4505835  PMID: 18326740
3.  Whole Exome Sequencing Reveals GUCY2D as a Major Gene Associated With Cone and Cone–Rod Dystrophy in Israel 
Purpose.
The Israeli population has a unique genetic make-up, with a high prevalence of consanguineous marriages and autosomal recessive diseases. In rod-dominated phenotypes, disease-causing genes and mutations that differ from those identified in other populations often are incurred. We used whole exome sequencing (WES) to identify genetic defects in Israeli families with cone-dominated retinal phenotypes.
Methods.
Clinical analysis included family history, detailed ocular examination, visual function testing, and retinal imaging. Whole exome sequencing, followed by segregation analysis, was performed in 6 cone-dominated retinopathy families in which prior mutation analysis did not reveal the causative gene. Based on the WES findings, we screened 106 additional families with cone-dominated phenotypes.
Results.
The WES analysis revealed mutations in known retinopathy genes in five of the six families: two pathogenic mutations in the GUCY2D gene in three families, and one each in CDHR1 and C8orf37. Targeted screening of additional cone-dominated families led to identification of GUCY2D mutations in four other families, which included two highly probable novel disease-causing variants.
Conclusions.
Our study suggested that GUCY2D is a major cause of autosomal dominant cone and cone–rod dystrophies in Israel; this is similar to other Caucasian populations and is in contrast with retinitis pigmentosa (primary rod disease), where the genetic make-up of the Israeli population is distinct from other ethnic groups. We also conclude that WES permits more comprehensive and rapid analyses that can be followed by targeted screens of larger samples to delineate the genetic structure of retinal disease in unique population cohorts.
Whole exome sequencing followed by targeted screening gives insight into the molecular genetic basis of cone-dominated retinal diseases in the Israeli population. The GUCY2D gene emerges as a major cause of autosomal dominant cone and cone–rod dystrophy, in similarity with other populations.
doi:10.1167/iovs.14-15647
PMCID: PMC4296770  PMID: 25515582
next generation sequencing; photoreceptor degeneration; inherited blindness
4.  The Transcription Factor GTF2IRD1 Regulates the Topology and Function of Photoreceptors by Modulating Photoreceptor Gene Expression across the Retina 
The Journal of Neuroscience  2014;34(46):15356-15368.
The mechanisms that specify photoreceptor cell-fate determination, especially as regards to short-wave-sensitive (S) versus medium-wave-sensitive (M) cone identity, and maintain their nature and function, are not fully understood. Here we report the importance of general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1) in maintaining M cone cell identity and function as well as rod function. In the mouse, GTF2IRD1 is expressed in cell-fate determined photoreceptors at postnatal day 10. GTF2IRD1 binds to enhancer and promoter regions in the mouse rhodopsin, M- and S-opsin genes, but regulates their expression differentially. Through interaction with the transcription factors CRX and thyroid hormone receptor β 2, it enhances M-opsin expression, whereas it suppresses S-opsin expression; and with CRX and NRL, it enhances rhodopsin expression. In an apparent paradox, although GTF2IRD1 is widely expressed in multiple cell types across the retina, knock-out of GTF2IRD1 alters the retinal expression of only a limited number of annotated genes. Interestingly, however, the null mutation leads to altered topology of cone opsin expression in the retina, with aberrant S-opsin overexpression and M-opsin underexpression in M cones. Gtf2ird1-null mice also demonstrate abnormal M cone and rod electrophysiological responses. These findings suggest an important role for GTF2IRD1 in regulating the level and topology of rod and cone gene expression, and in maintaining normal retinal function.
doi:10.1523/JNEUROSCI.2089-14.2014
PMCID: PMC4228138  PMID: 25392503
gene expression; opsin; photoreceptor; retina; transcription factor
5.  Clinical and Genetic Factors Associated with Progression of Geographic Atrophy Lesions in Age-Related Macular Degeneration 
PLoS ONE  2015;10(5):e0126636.
Worldwide, age-related macular degeneration (AMD) is a serious threat to vision loss in individuals over 50 years of age with a pooled prevalence of approximately 9%. For 2020, the number of people afflicted with this condition is estimated to reach 200 million. While AMD lesions presenting as geographic atrophy (GA) show high inter-individual variability, only little is known about prognostic factors. Here, we aimed to elucidate the contribution of clinical, demographic and genetic factors on GA progression. Analyzing the currently largest dataset on GA lesion growth (N = 388), our findings suggest a significant and independent contribution of three factors on GA lesion growth including at least two genetic factors (ARMS2_rs10490924 [P < 0.00088] and C3_rs2230199 [P < 0.00015]) as well as one clinical component (presence of GA in the fellow eye [P < 0.00023]). These correlations jointly explain up to 7.2% of the observed inter-individual variance in GA lesion progression and should be considered in strategy planning of interventional clinical trials aimed at evaluating novel treatment options in advanced GA due to AMD.
doi:10.1371/journal.pone.0126636
PMCID: PMC4427438  PMID: 25962167
6.  The transcription-splicing protein NonO/p54nrb and three NonO-interacting proteins bind to distal enhancer region and augment rhodopsin expression 
Human Molecular Genetics  2013;23(8):2132-2144.
Phototransduction machinery in vertebrate photoreceptors is contained within the membrane discs of outer segments. Daily renewal of 10% of photoreceptor outer segments requires stringent control of gene expression. Rhodopsin constitutes over 90% of the protein in rod discs, and its altered expression or transport is associated with photoreceptor dysfunction and/or death. Two cis-regulatory sequences have been identified upstream of the rhodopsin transcription start site. While the proximal promoter binds to specific transcription factors, including NRL and CRX, the rhodopsin enhancer region (RER) reportedly contributes to precise and high-level expression of rhodopsin in vivo. Here, we report the identification of RER-bound proteins by mass spectrometry. We validate the binding of NonO (p54nrb), a protein implicated in coupling transcription to splicing, and three NonO-interacting proteins—hnRNP M, Ywhaz and Ppp1ca. NonO and its interactors can activate rhodopsin promoter in HEK293 cells and function synergistically with NRL and CRX. DNA-binding domain of NonO is critical for rhodopsin promoter activation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis demonstrates high occupancy of NonO at rhodopsin and a subset of phototransduction genes. Furthermore, shRNA knockdown of NonO in mouse retina leads to loss of rhodopsin expression and rod cell death, which can be partially rescued by a C-terminal NonO construct. RNA-seq analysis of the NonO shRNA-treated retina revealed splicing defects and altered expression of genes, specifically those associated with phototransduction. Our studies identify an important contribution of NonO and its interacting modulator proteins in enhancing rod-specific gene expression and controlling rod homeostasis.
doi:10.1093/hmg/ddt609
PMCID: PMC3959818  PMID: 24301678
7.  Deletion of Aryl Hydrocarbon Receptor AHR in Mice Leads to Subretinal Accumulation of Microglia and RPE Atrophy 
Purpose.
The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis.
Methods.
RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr−/− mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours.
Results.
In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr−/− mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr−/− retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr−/− mice displayed reduced ERG c-wave amplitudes.
Conclusions.
The Ahr−/− mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr−/− mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention.
We show that aryl hydrocarbon receptor (AHR) is expressed in the neural retina in addition to RPE and that its loss results in subretinal microglia and RPE atrophy, some of the phenotypes observed in AMD. Thus, AHR mediates a protective mechanism in ocular tissues and is a candidate target for intervention.
doi:10.1167/iovs.14-15091
PMCID: PMC4176417  PMID: 25159211
orphan nuclear receptor; retinal degeneration; RPE atrophy; subretinal deposits; microglia
8.  Biology and therapy of inherited retinal degenerative disease: insights from mouse models 
Disease Models & Mechanisms  2015;8(2):109-129.
Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases.
doi:10.1242/dmm.017913
PMCID: PMC4314777  PMID: 25650393
Mouse mutants; Photoreceptor; Retinal development; Retinal disease
9.  Genome-wide association study and meta-analysis of intraocular pressure 
Human genetics  2013;133(1):41-57.
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.
doi:10.1007/s00439-013-1349-5
PMCID: PMC3982323  PMID: 24002674
10.  Hypomethylation of the IL17RC Promoter in Peripheral Blood Leukocytes is Not A Hallmark of Age-Related Macular Degeneration 
Cell reports  2013;5(6):1527-1535.
SUMMARY
Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely-accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive QPCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application.
doi:10.1016/j.celrep.2013.11.042
PMCID: PMC3926096  PMID: 24373284
11.  Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis 
Nature communications  2014;5:4207.
The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a-/- mouse that recapitulates features of Meckel syndrome including embryonic lethality and multi-organ defects. Cilia are absent in Cc2d2a-/- embryonic node and other somatic tissues; disruption of cilia-dependent Shh signaling appears to underlie exencephaly in mutant embryos. The Cc2d2a-/- mouse embryonic fibroblasts (MEFs) lack cilia though mother centriole and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a-/- MEFs, subdistal appendages are lacking or abnormal by transmission-EM. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.
doi:10.1038/ncomms5207
PMCID: PMC4096663  PMID: 24947469
12.  Age-Related Macular Degeneration: Genetics and Biology Coming Together 
Genetic and genomic studies have enhanced our understanding of complex neurodegenerative diseases that exert a devastating impact on individuals and society. One such disease, age-related macular degeneration (AMD), is a major cause of progressive and debilitating visual impairment. Since the pioneering discovery in 2005 of complement factor H (CFH) as a major AMD susceptibility gene, extensive investigations have confirmed 19 additional genetic risk loci, and more are anticipated. In addition to common variants identified by now-conventional genome-wide association studies, targeted genomic sequencing and exome-chip analyses are uncovering rare variant alleles of high impact. Here, we provide a critical review of the ongoing genetic studies and of common and rare risk variants at a total of 20 susceptibility loci, which together explain 40–60% of the disease heritability but provide limited power for diagnostic testing of disease risk. Identification of these susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.
doi:10.1146/annurev-genom-090413-025610
PMCID: PMC4217162  PMID: 24773320
complex disease; genetic susceptibility; neurodegeneration; retina; blindness
13.  Genetic architecture of retinal and macular degenerative diseases: the promise and challenges of next-generation sequencing 
Genome Medicine  2013;5(10):84.
Inherited retinal degenerative diseases (RDDs) display wide variation in their mode of inheritance, underlying genetic defects, age of onset, and phenotypic severity. Molecular mechanisms have not been delineated for many retinal diseases, and treatment options are limited. In most instances, genotype-phenotype correlations have not been elucidated because of extensive clinical and genetic heterogeneity. Next-generation sequencing (NGS) methods, including exome, genome, transcriptome and epigenome sequencing, provide novel avenues towards achieving comprehensive understanding of the genetic architecture of RDDs. Whole-exome sequencing (WES) has already revealed several new RDD genes, whereas RNA-Seq and ChIP-Seq analyses are expected to uncover novel aspects of gene regulation and biological networks that are involved in retinal development, aging and disease. In this review, we focus on the genetic characterization of retinal and macular degeneration using NGS technology and discuss the basic framework for further investigations. We also examine the challenges of NGS application in clinical diagnosis and management.
doi:10.1186/gm488
PMCID: PMC4066589  PMID: 24112618
14.  Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation 
Neurobiology of aging  2013;34(10):2310-2321.
Microglia, the resident immune cell of the central nervous system (CNS), are thought to contribute to the pathogenesis of age-related neurodegenerative disorders. It has been hypothesized that microglia undergo age-related changes in gene expression patterns that give rise to pathogenic phenotypes. We compared the gene expression profiles in microglia isolated ex vivo from the mouse retinas of ages ranging from early adulthood to late senescence. We discovered that microglial gene expression demonstrated progressive change with increasing age and involved genes that regulate microglial supportive functions and immune activation. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling demonstrated age-related change. In particular, expression levels of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent microglia may contribute to complement dysregulation during disease pathogenesis. Taken together, senescent microglia demonstrate age-related gene expression changes capable of altering their constitutive support functions and regulation of their activation status in ways relating to neuroinflammation and neurodegeneration in the CNS.
doi:10.1016/j.neurobiolaging.2013.03.022
PMCID: PMC3706521  PMID: 23608111
aging; microglia; retina; microarray; gene expression; complement; activation; angiogenesis; neurotrophic factors; senescence
15.  Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a 
Biology Open  2014;3(9):861-870.
ABSTRACT
Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six “core PCP” components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. We recently demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, we generated three mutant alleles in mice. We show that the complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting us to explore an association of Prickle1 with the Wnt pathway. We show that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. Our studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome.
doi:10.1242/bio.20148375
PMCID: PMC4163663  PMID: 25190059
Planar cell polarity; Development; Morphogenesis; Organogenesis; Conditional mouse mutants
16.  Ancestry Estimation and Control of Population Stratification for Sequence-based Association Studies 
Nature genetics  2014;46(4):409-415.
Knowledge of individual ancestry is important for genetic association studies where population structure leads to false positive signals. Estimating individual ancestry with targeted sequence data, which constitutes the bulk of current sequence datasets, is challenging. Here, we propose a new method for accurate estimation of genetic ancestry. Our method skips genotype calling and directly analyzes sequence reads. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry with whole genome shotgun coverage as low as 0.001X. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1X. At an even finer-scale, the method improves discrimination between exome-sequenced participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and reduce the risk of spurious findings due to population structure.
doi:10.1038/ng.2924
PMCID: PMC4084909  PMID: 24633160
17.  Prickle1 is expressed in distinct cell populations of the central nervous system and contributes to neuronal morphogenesis 
Human Molecular Genetics  2013;22(11):2234-2246.
Development of axons and dendrites constitutes a critical event in neuronal maturation and seems to require signaling through the planar cell polarity (PCP) pathway. Mutations in components of the PCP pathway lead to a spectrum of neurological phenotypes and disorders. For example, a missense mutation in Prickle 1 (Pk1) is associated with progressive myoclonus epilepsy (PME) in humans, and its reduced gene dosage increases sensitivity to induced seizure in mice. In an effort to unravel the role of the PCP pathway in mammalian neuronal development, we examined the expression of Pk1 in the central nervous system (CNS) using in situ hybridization (ISH) in combination with a genetic knock-in approach. We show that Pk1 transcripts are detected in the postmitotic cells of the subplate and cortical plate during mid- and late stages of cortical neurogenesis. In adult brain, Pk1 is expressed in distinct neuronal and glial cell populations, with dynamic formation of dendrites and glial processes during development. Of all the cell types in the mature retina, the highest expression of Pk1 is detected in cholinergic amacrine neurons. Knockdown of Pk1 by shRNA or dominant-negative constructs causes reduced axonal and dendritic extension in hippocampal neurons. Similarly, Pk1 knockdown in neonatal retina leads to defects in inner and outer segments and axon terminals of photoreceptors. Our studies implicate Pk1 function in axonal-dendritic development associated with the maturation of CNS neurons.
doi:10.1093/hmg/ddt075
PMCID: PMC3652420  PMID: 23420014
18.  Development and Plasticity of Outer Retinal Circuitry Following Genetic Removal of Horizontal Cells 
The Journal of Neuroscience  2013;33(45):17847-17862.
The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.
doi:10.1523/JNEUROSCI.1373-13.2013
PMCID: PMC3818557  PMID: 24198374
19.  Identification of a Rare Coding Variant in Complement 3 Associated with Age-related Macular Degeneration 
Nature genetics  2013;45(11):10.1038/ng.2758.
Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry matched controls revealed two large-effect rare variants; previously described R1210C in the CFH gene (fcase = 0.51%, fcontrol = 0.02%, OR = 23.11), and newly identified K155Q in the C3 gene (fcase = 1.06%, fcontrol = 0.39%, OR = 2.68). The variants suggest decreased inhibition of C3 by Factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology.
doi:10.1038/ng.2758
PMCID: PMC3812337  PMID: 24036949
20.  CP110 Suppresses Primary Cilia Formation through its Interaction with CEP290, a Protein Deficient in Human Ciliary Disease 
Developmental cell  2008;15(2):187-197.
Primary cilia are non-motile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290-a protein whose deficiency is implicated in human ciliary disease-in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and this function is antagonized by CP110.
doi:10.1016/j.devcel.2008.07.004
PMCID: PMC3987787  PMID: 18694559
CP110; CEP290; Rab8a; ciliogenesis; primary cilia
21.  Natural History of Cone Disease in the Murine Model of Leber Congenital Amaurosis Due to CEP290 Mutation: Determining the Timing and Expectation of Therapy 
PLoS ONE  2014;9(3):e92928.
Background
Mutations in the CEP290 (cilia-centrosomal protein 290 kDa) gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, rd16;Nrl−/−, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.
Methods
Rd16;Nrl−/− mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA (n = 15; ages 7–48) were compared with those of the model.
Results
CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea (despite severe visual loss); the extent of this island declined slowly with age. The rd16;Nrl−/− model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant Nrl−/− mice, UV- and M-cone ERGs of rd16;Nrl−/− were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age.
Conclusions
The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both CEP290-LCA patients and the rd16;Nrl−/− murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG.
doi:10.1371/journal.pone.0092928
PMCID: PMC3966841  PMID: 24671090
22.  OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness 
Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.
doi:10.1172/JCI72722
PMCID: PMC3904630  PMID: 24382353
23.  Ablation of the X-Linked Retinitis Pigmentosa 2 (Rp2) Gene in Mice Results in Opsin Mislocalization and Photoreceptor Degeneration 
Purpose.
Mutations in the RP2 gene are associated with 10% to 15% of X-linked retinitis pigmentosa (XLRP), a debilitating disorder characterized by the degeneration of retinal rod and cone photoreceptors. The molecular mechanism of pathogenesis of photoreceptor degeneration in XLRP-RP2 has not been elucidated, and no treatment is currently available. This study was undertaken to investigate the pathogenesis of RP2-associated retinal degeneration.
Methods.
We introduced loxP sites that flank exon 2, a mutational hotspot in XLRP-RP2, in the mouse Rp2 gene. We then produced Rp2-null allele using transgenic mice that expressed Cre-recombinase under control of the ubiquitous CAG promoter. Electroretinography (ERG), histology, light microscopy, transmission electron microscopy, and immunofluorescence microscopy were performed to ascertain the effect of ablation of Rp2 on photoreceptor development, function, and protein trafficking.
Results.
Although no gross abnormalities were detected in the Rp2null mice, photopic (cone) and scotopic (rod) function as measured by ERG showed a gradual decline starting as early as 1 month of age. We also detected slow progressive degeneration of the photoreceptor membrane discs in the mutant retina. These defects were associated with mislocalization of cone opsins to the nuclear and synaptic layers and reduced rhodopsin content in the outer segment of mutant retina prior to the onset of photoreceptor degeneration.
Conclusions.
Our studies suggest that RP2 contributes to the maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in XLRP caused by RP2 mutations. The Rp2null mice should serve as a useful preclinical model for testing gene- and cell-based therapies.
This study reports generation and characterization of a mouse model of Rp2-mediated retinal degeneration and shows that cone opsin mislocalization is an early step in the pathogenesis of associated disease.
doi:10.1167/iovs.13-12140
PMCID: PMC3700388  PMID: 23745007
Rp2; retina; photoreceptor
24.  Genetic studies of Age-related macular degeneration: lessons, challenges and opportunities for disease management 
Ophthalmology  2012;119(12):2526-2536.
Age-related macular degeneration (AMD) is a common cause of visual impairment in individuals over 55 years of age worldwide. The varying clinical phenotypes of AMD result from contributions of genetic, epigenetic and non-genetic (environmental) factors. Genetic studies of AMD have come of age as a direct result of tremendous gains from human genome project, genomewide association studies and identification of numerous susceptibility loci. These findings have implicated immune response, high-density lipoprotein cholesterol metabolism, extracellular matrix, and angiogenesis signaling pathways in disease pathophysiology. Here, we address how the wealth of genetic findings in AMD is expected to impact the practice of medicine, providing opportunities for improved risk assessment, molecular diagnosis, preventive and therapeutic intervention. We propose that the potential of using genetic variants for monitoring treatment response (pharmacogenetics) may usher a new era of personalized medicine in the clinical management of AMD.
doi:10.1016/j.ophtha.2012.06.042
PMCID: PMC3514599  PMID: 23009893
Retinal neurodegeneration; Complex disease; Genomewide association; Risk factors; Genetic diagnosis; Pharmacogenetics; Clinical management
25.  An isoform of retinoid-related orphan receptor β directs differentiation of retinal amacrine and horizontal interneurons 
Nature communications  2013;4:1813.
Amacrine and horizontal interneurons integrate visual information as it is relayed through the retina from the photoreceptors to the ganglion cells. The early steps that generate these interneuron networks remain unclear. Here we show that a distinct RORβ1 isoform encoded by the retinoid-related orphan nuclear receptor β gene (Rorb) is critical for both amacrine and horizontal cell differentiation in mice. A fluorescent protein cassette targeted into Rorb revealed RORβ1 as a novel marker of immature amacrine and horizontal cells and of undifferentiated, dividing progenitor cells. RORβ1-deficient mice lose expression of pancreas-specific transcription factor 1a (Ptf1a) but retain forkhead box n4 factor (Foxn4), two early-acting factors necessary for amacrine and horizontal cell generation. RORβ1 and Foxn4 synergistically induce Ptf1a expression, suggesting a central role for RORβ1 in a transcriptional hierarchy that directs this interneuron differentiation pathway. Moreover, ectopic RORβ1 expression in neonatal retina promotes amacrine cell differentiation.
doi:10.1038/ncomms2793
PMCID: PMC3671912  PMID: 23652001
orphan nuclear receptor; forkhead box transcription factor Foxn4; basic helix-loop-helix factor Ptf1a; neurogenesis; inhibitory interneuron

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