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1.  Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy 
Human Molecular Genetics  2015;24(22):6446-6458.
Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development.
doi:10.1093/hmg/ddv354
PMCID: PMC4626763  PMID: 26358772
2.  Stem cells with a view: a look inside a retinal ciliopathy 
doi:10.21037/sci.2016.09.19
PMCID: PMC5104580  PMID: 27868044
3.  Loss of Retinitis Pigmentosa 2 (RP2) protein affects cone photoreceptor sensory cilium elongation in mice 
Cytoskeleton (Hoboken, N.J.)  2015;72(9):447-454.
Degeneration of photoreceptors (rods and cones) results in blindness. As we rely almost entirely on our daytime vision mediated by the cones, it is the loss of these photoreceptors that results in legal blindness and poor quality of life. Cone dysfunction is usually observed due to two mechanisms: non cell-autonomous due to the secondary effect of rod death if the causative gene is specifically expressed in rods, and cell autonomous, if the mutation is in a cone-specific gene. However, it is difficult to dissect cone autonomous effect of mutations in the genes that are expressed in both rods and cones. Here we report a property of murine cone photoreceptors, which is a cone-autonomous effect of the genetic perturbation of the retinitis pigmentosa 2 (Rp2) gene mutated in human X-linked RP. Constitutive loss of Rp2 results in abnormal extension of the cone outer segment (COS). This effect is phenocopied when the Rp2 gene is ablated specifically in cones but not when ablated in rods. Furthermore, the elongated COS exhibits abnormal ultrastructure with disorganized lamellae. Additionally, elongation of both the OS membrane and the microtubule cytoskeleton was observed in the absence of RP2. Taken together, our studies identify a cone morphological defect in retinal degeneration due to ablation of RP2 and will assist in understanding cone-autonomous responses during disease and develop targeted therapies.
doi:10.1002/cm.21255
PMCID: PMC4715527  PMID: 26383048
mouse model; photoreceptor; retina; cilia
4.  Advances in Gene Therapy for Diseases of the Eye 
Human Gene Therapy  2016;27(8):563-579.
Over the last few years, huge progress has been made with regard to the understanding of molecular mechanisms underlying the pathogenesis of neurodegenerative diseases of the eye. Such knowledge has led to the development of gene therapy approaches to treat these devastating disorders. Challenges regarding the efficacy and efficiency of therapeutic gene delivery have driven the development of novel therapeutic approaches, which continue to evolve the field of ocular gene therapy. In this review article, we will discuss the evolution of preclinical and clinical strategies that have improved gene therapy in the eye, showing that treatment of vision loss has a bright future.
doi:10.1089/hum.2016.040
PMCID: PMC4991575  PMID: 27178388
5.  The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo 
Biology Open  2016;5(4):424-428.
ABSTRACT
Mutations in RPGRORF15 (retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGRORF15 (1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGRORF15 is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGRC2 domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGRORF15 is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants of Rpgr, which do not express RPGRORF15 due to disease-causing mutations in exon ORF15. Our results indicate that RPGRORF15 is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGRORF15 in photoreceptor cilia.
Summary: This study shows that the mutational hotspot of ciliary protein RPGRORF15, commonly associated with severe blindness, is posttranslationally glutamylated at its C-terminus and is a target of GT335.
doi:10.1242/bio.016816
PMCID: PMC4890669  PMID: 26941104
Retina; RPGR; Cilia; Glutamylation; GT335
6.  Photoreceptor Sensory Cilium: Traversing the Ciliary Gate 
Cells  2015;4(4):674-686.
Cilia are antenna-like extensions of the plasma membrane found in nearly all cell types. In the retina of the eye, photoreceptors develop unique sensory cilia. Not much was known about the mechanisms underlying the formation and function of photoreceptor cilia, largely because of technical limitations and the specific structural and functional modifications that cannot be modeled in vitro. With recent advances in microscopy techniques and molecular and biochemical approaches, we are now beginning to understand the molecular basis of photoreceptor ciliary architecture, ciliary function and its involvement in human diseases. Here, I will discuss the studies that have revealed new knowledge of how photoreceptor cilia regulate their identity and function while coping with high metabolic and trafficking demands associated with processing light signal.
doi:10.3390/cells4040674
PMCID: PMC4695852  PMID: 26501325
cilia; flagella; ciliopathies; retina; retinopathies; transition zone; protein trafficking
7.  Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition 
Scientific Reports  2015;5:11137.
Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgrko retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgrko PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgrko PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.
doi:10.1038/srep11137
PMCID: PMC4463945  PMID: 26068394
8.  Loss of Raf-1 Kinase Inhibitory Protein Delays Early-Onset Severe Retinal Ciliopathy in Cep290rd16 Mouse 
Purpose.
Mutations in the cilia-centrosomal protein of centrosomal protein of 290 kDa (CEP290) result in severe ciliopathies, including autosomal recessive early onset childhood blindness disorder Leber congenital amaurosis (LCA). The Cep290rd16 (retinal degeneration 16) mouse model of CEP290-LCA exhibits accumulation of CEP290-interacting protein Raf-1 kinase inhibitory protein (RKIP) prior to onset of retinal degeneration (by postnatal day P14). We hypothesized that reducing RKIP levels in the Cep290rd16 mouse will delay or improve retinal phenotype.
Methods.
We generated double mutant mice by combining the Cep290rd16 and Rkipko alleles (Cep290rd16:Rkip+/ko and Cep290rd16:Rkipko/ko). Retinal function was assessed by ERG and retinal morphology and protein trafficking were assessed by histology, transmission electron microscopy (TEM), and immunofluorescence analysis. Cell death was examined by apoptosis.
Results.
Prior to testing our hypothesis, we examined ERG and retinal morphology of Rkipko/ko mice and did not find any detectable differences compared with wild-type mice. The Cep290rd16:Rkip+/ko mice exhibited similar retinopathy as Cep290rd16; however, Cep290rd16: Rkipko/ko double knockout mice demonstrated a substantial improvement (>9-fold) in photoreceptor function and structure at P18 as of Cep290rd16 mice. We consistently detected transient preservation of photoreceptors at P18 and polarized trafficking of opsins to sensory cilia in the double mutant mice; however, retinal degeneration ensued by P30.
Conclusions.
Our studies implicate CEP290-RKIP pathway in CEP290-retinal degeneration and suggest that targeting RKIP levels can delay photoreceptor degeneration, assisting in extending the time-window for treating such rapidly progressing blindness disorder.
This manuscript shows that CEP290-associated fast progressing early-onset retinal phenotype in rd16 mouse can be delayed by reducing the levels of one of its interacting proteins, RKIP. The work provides a platform to design intermediates that can be supplemented with other therapeutic strategies.
doi:10.1167/iovs.14-14954
PMCID: PMC4165369  PMID: 25125607
CEP290; RKIP; retina; photoreceptor degeneration; blindness
9.  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
11.  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
12.  CILIARY SIGNALING CASCADES IN PHOTORECEPTORS 
Vision research  2012;75:112-116.
For being a polarized neuron and having a sensory cilium, photoreceptors attract remarkable attention. This is due their highly polarized structure and active visual signal transduction cascades and for the enrichment of complex networks of proteins in the cilium. Structural and functional maintenance of the photoreceptor sensory cilium, also called outer segment, ensures that light signal is received and relayed appropriately to the brain. Any perturbations in the protein content of the outer segment result in photoreceptor dysfunction, degeneration and eventually, blindness. This review focuses on the importance of photoreceptor sensory cilium to carry out signal transduction cascade for vision.
doi:10.1016/j.visres.2012.08.007
PMCID: PMC3724345  PMID: 22921640
13.  Ciliary Transition Zone (TZ) Proteins RPGR and CEP290: Role in Photoreceptor Cilia and Degenerative Diseases 
Introduction
Primary cilia are microtubule-based extensions of the plasma membrane in nearly all cell types. In vertebrate photoreceptors, the sensory cilium develops as outer segment (OS) that contains the photopigment Rhodopsin and other proteins necessary for phototransduction. The distinct composition of proteins and lipids in the OS membrane is maintained by the selective barrier located at the border between the basal body and the ciliary compartment, called the Transition Zone (TZ).
Areas covered
In this review, we will discuss the identification and function of two ciliary TZ proteins, RPGR (retinitis pigmentosa GTPase regulator) and CEP290. Mutations in these proteins account for a majority of retinopathies due to ciliary dysfunction. We will also discuss the potential of such information in designing therapeutic approaches to treat cilia-dependent photoreceptor degenerative diseases.
Expert opinion
RPGR and CEP290 perform overlapping yet distinct functions in regulating trafficking of cargo via the TZ of photoreceptors. While RPGR modulates the trafficking by acting as a GEF for the small GTPase RAB8A, CEP290 may be involved in maintaining the polarized distribution of proteins in the OS by modulating intracellular levels of selected proteins involved in inhibiting OS formation.
doi:10.1517/14728222.2012.680956
PMCID: PMC3724338  PMID: 22563985
Ciliopathies; cilia; retina; retinitis pigmentosa; retinal degeneration; Leber congenital amaurosis; Ciliary trafficking; connecting cilium; transition zone
14.  Multiprotein Complexes of Retinitis Pigmentosa GTPase Regulator (RPGR), a Ciliary Protein Mutated in X-Linked Retinitis Pigmentosa (XLRP) 
Mutations in Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of X-linked Retinitis Pigmentosa (XLRP). The RPGR gene undergoes extensive alternative splicing and encodes for distinct protein isoforms in the retina. Extensive studies using isoform-specific antibodies and mouse mutants have revealed that RPGR predominantly localizes to the transition zone to primary cilia and associates with selected ciliary and microtubule-associated assemblies in photoreceptors. In this chapter, we have summarized recent advances on understanding the role of RPGR in photoreceptor protein trafficking. We also provide new evidence that suggests the existence of discrete RPGR multiprotein complexes in photoreceptors. Piecing together the RPGR-interactome in different subcellular compartments should provide critical insights into the role of alternative RPGR isoforms in associated orphan and syndromic retinal degenerative diseases.
doi:10.1007/978-1-4419-1399-9_13
PMCID: PMC3464500  PMID: 20238008
15.  RPGR-containing protein complexes in syndromic and non-syndromic retinal degeneration due to ciliary dysfunction 
Journal of genetics  2009;88(4):399-407.
Dysfunction of primary cilia due to mutations in cilia-centrosomal proteins is associated with pleiotropic disorders. The primary (or sensory) cilium of photoreceptors mediates polarized trafficking of proteins for efficient phototransduction. Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein mutated in >70% of X-linked RP cases and 10%–20% of simplex RP males. Accumulating evidence indicates that RPGR may facilitate the orchestration of multiple ciliary protein complexes. Disruption of these complexes due to mutations in component proteins is an underlying cause of associated photoreceptor degeneration. Here, we highlight the recent developments in understanding the mechanism of cilia-dependent photoreceptor degeneration due to mutations in RPGR and RPGR-interacting proteins in severe genetic diseases, including retinitis pigmentosa, Leber congenital amaurosis (LCA), Joubert syndrome, and Senior–Loken syndrome, and explore the physiological relevance of photoreceptor ciliary protein complexes.
PMCID: PMC3464916  PMID: 20090203
primary cilia; centrosome; transition zone; ciliopathies; photoreceptor; retinal degeneration; retina; RPGR; RP2; CEP290; RPGRIP1L; NPHP
17.  THE RP2 PHENOTYPE AND PATHOGENETIC CORRELATIONS IN X-LINKED RETINITIS PIGMENTOSA 
Archives of Ophthalmology  2010;128(7):915-923.
Objective
To assess the phenotype of X-linked retinitis pigmentosa (XLRP) patients with RP2 mutations and correlate the findings with their genotype.
Clinical Relevance
An identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.
Methods
Over 600 XLRP patients and carriers were screened during a ten-year period for mutations in the RP2 gene. Twenty-five RP2 patients were evaluated clinically with standardized electroretinography (ERG), Goldmann visual fields, and ocular examinationsl. In addition, well documented cases from the literature were used to augment genotype-phenotype correlations.
Results
In our male cohort under the age of 12 years: 10/11 (91%) patients had macular involvement and 10/11 (91%) had best corrected visual acuities worse than 20/50. Two males from different families (ages 8 and 12) displayed a choroideremia-like fundus, and 9/11( 82%) of male patients were myopic with a mean error of −7.97D. Of patients with ERG data, 9/10 (90%) demonstrated severe rod-cone dysfunction. All three female carriers had macular atrophy in one or both eyes and were myopic (mean −6.23 D). We identified four novel RP2 mutations. All nine nonsense and five of seven missense mutations (71%) resulted in severe clinical presentations.
Conclusion
Screening of the RP2 gene should be prioritized in patients less than 16 years of age characterized by X-linked inheritance, decreased BCVA (e.g.,>20/40), high myopia, and early-onset macular atrophy. We also suggest that patients exhibiting a choroideremia-like fundus appearance who do not have disease-causing mutations in the choroideremia gene (CHM) be screened for variations in RP2. We believe that alterations in function play a significant role in RP2-associated disease pathogenesis.
doi:10.1001/archophthalmol.2010.122
PMCID: PMC3392190  PMID: 20625056
18.  Expression and Functional Roles of Caspase-5 in Inflammatory Responses of Human Retinal Pigment Epithelial Cells 
Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation of caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.
Purpose.
To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells.
Methods.
Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis.
Results.
Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1–specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α–induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate.
Conclusions.
Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.
doi:10.1167/iovs.11-7570
PMCID: PMC3230287  PMID: 21969293
19.  Rd9 Is a Naturally Occurring Mouse Model of a Common Form of Retinitis Pigmentosa Caused by Mutations in RPGR-ORF15 
PLoS ONE  2012;7(5):e35865.
Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.
doi:10.1371/journal.pone.0035865
PMCID: PMC3341386  PMID: 22563472
20.  OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome 
Human Molecular Genetics  2012;21(15):3333-3344.
Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.
doi:10.1093/hmg/dds163
PMCID: PMC3392109  PMID: 22543976
21.  Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis  
The Journal of Clinical Investigation  2012;122(4):1233-1245.
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
doi:10.1172/JCI60981
PMCID: PMC3314468  PMID: 22446187
22.  TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein 
Human Molecular Genetics  2010;20(5):975-987.
We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.
doi:10.1093/hmg/ddq543
PMCID: PMC3033188  PMID: 21159800
23.  The retinitis pigmentosa protein RP2 interacts with polycystin 2 and regulates cilia-mediated vertebrate development 
Human Molecular Genetics  2010;19(22):4330-4344.
Ciliopathies represent a growing group of human genetic diseases whose etiology lies in defects in ciliogenesis or ciliary function. Given the established entity of renal–retinal ciliopathies, we have been examining the role of cilia-localized proteins mutated in retinitis pigmentosa (RP) in regulating renal ciliogenesis or cilia-dependent signaling cascades. Specifically, this study examines the role of the RP2 gene product with an emphasis on renal and vertebrate development. We demonstrate that in renal epithelia, RP2 localizes to the primary cilium through dual acylation of the amino-terminus. We also show that RP2 forms a calcium-sensitive complex with the autosomal dominant polycystic kidney disease protein polycystin 2. Ablation of RP2 by shRNA promotes swelling of the cilia tip that may be a result of aberrant trafficking of polycystin 2 and other ciliary proteins. Morpholino-mediated repression of RP2 expression in zebrafish results in multiple developmental defects that have been previously associated with ciliary dysfunction, such as hydrocephalus, kidney cysts and situs inversus. Finally, we demonstrate that, in addition to our observed physical interaction between RP2 and polycystin 2, dual morpholino-mediated knockdown of polycystin 2 and RP2 results in enhanced situs inversus, indicating that these two genes also regulate a common developmental process. This work suggests that RP2 may be an important regulator of ciliary function through its association with polycystin 2 and provides evidence of a further link between retinal and renal cilia function.
doi:10.1093/hmg/ddq355
PMCID: PMC2957320  PMID: 20729296
24.  XIAP Therapy Increases Survival of Transplanted Rod Precursors in a Degenerating Host Retina 
Survival of integrated cells decreases with time after transplantation but can be significantly increased with XIAP antiapoptotic therapy. Preventing programmed cell death through XIAP therapy may be an important component of future therapeutic retinal cell transplantation strategies.
Purpose.
To assess the survival of rod precursor cells transplanted into the Rd9 mouse, a model of X-linked retinal degeneration, and the effect of antiapoptotic therapy with X-linked inhibitor of apoptosis (XIAP) on preventing cell loss.
Methods.
Dissociated retinal cells from P4 Nrlp-GFP mice were transplanted into the subretinal space of 2-, 5-, and 8-month-old Rd9 mice. Histology, immunohistochemistry, and quantification of integrated cells were performed every month for up to 3 months after transplantation. XIAP delivery to donor cells was accomplished by transfection with adenoassociated virus (AAV-XIAP). Intraretinal activation of immune modulators was assessed using a quantitative real-time polymerase chain reaction-based immune response array.
Results.
GFP-positive rod precursors were able to integrate into the outer nuclear layer (ONL) of the Rd9 retina. Transplanted cells underwent morphologic differentiation with the formation of inner and outer segments and synaptic projections to bipolar cells. Integration of donor cells into the ONL increased as a function of host age at the time of transplantation. The number of integrated cells was maximal at 1 month after transplantation and then decreased with time. Survival of integrated cells was significantly increased when donor cells were pretreated with AAV-XIAP. We did not detect any donor cell-specific activation of inflammation within the host retina.
Conclusions.
Survival of integrated cells decreases with time after transplantation but can be significantly increased with XIAP antiapoptotic therapy. Preventing programmed cell death through XIAP therapy may be an important component of future therapeutic retinal cell transplantation strategies.
doi:10.1167/iovs.10-5998
PMCID: PMC3101692  PMID: 20926819
25.  Functional Analysis of Retinitis Pigmentosa 2 (RP2) Protein Reveals Variable Pathogenic Potential of Disease-Associated Missense Variants 
PLoS ONE  2011;6(6):e21379.
Genetic mutations are frequently associated with diverse phenotypic consequences, which limits the interpretation of the consequence of a variation in patients. Mutations in the retinitis pigmentosa 2 (RP2) gene are associated with X-linked RP, which is a phenotypically heterogenic form of retinal degeneration. The purpose of this study was to assess the functional consequence of disease-associated mutations in the RP2 gene using an in vivo assay. Morpholino-mediated depletion of rp2 in zebrafish resulted in perturbations in photoreceptor development and microphthalmia (small eye). Ultrastructural and immunofluorescence analyses revealed defective photoreceptor outer segment development and lack of expression of photoreceptor-specific proteins. The retinopathy phenotype could be rescued by expressing the wild-type human RP2 protein. Notably, the tested RP2 mutants exhibited variable degrees of rescue of rod versus cone photoreceptor development as well as microphthalmia. Our results suggest that RP2 plays a key role in photoreceptor development and maintenance in zebrafish and that the clinical heterogeneity associated with RP2 mutations may, in part, result from its potentially distinct functional relevance in rod versus cone photoreceptors.
doi:10.1371/journal.pone.0021379
PMCID: PMC3124502  PMID: 21738648

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