An FRMD4B variant suppresses dysplastic photoreceptor lesions in models of enhanced S-cone syndrome and of Nrl deficiency.

Photoreceptor dysplasia, characterized by formation of folds and (pseudo-)rosettes in the outer retina, is associated with loss of functional nuclear receptor subfamily 2 group E member 3 (NR2E3) and neural retina leucine-zipper (NRL) in both humans and mice. A sensitized chemical mutagenesis study to identify genetic modifiers that suppress photoreceptor dysplasia in Nr2e3rd7mutant mice identified line Tvrm222, which exhibits a normal fundus appearance in the presence of the rd7 mutation. The Tvrm222 modifier of Nr2e3rd7/rd7 was localized to Chromosome 6 and identified as a missense mutation in the FERM domain containing 4B (Frmd4b) gene. The variant is predicted to cause the substitution of a serine residue 938 with proline (S938P). The Frmd4bTvrm222 allele was also found to suppress outer nuclear layer (ONL) rosettes in Nrl-/- mice. Fragmentation of the external limiting membrane (ELM), normally observed in rd7 and Nrl-/-mouse retinas, was absent in the presence of the Frmd4bTvrm222 allele. FRMD4B, a binding partner of cytohesin 3, is proposed to participate in cell junction remodeling. Its biological function in photoreceptor dysplasia has not been previously examined. In vitro experiments showed that the FRMD4B938P variant fails to be efficiently recruited to the cell surface upon insulin stimulation. In addition, we found a reduction in protein kinase B phosphorylation and increased levels of cell junction proteins, Catenin beta 1 and tight junction protein 1, associated with the cell membrane in Tvrm222 retinas. Taken together, this study reveals a critical role of FRMD4B in maintaining ELM integrity and in rescuing morphological abnormalities of the ONL in photoreceptor dysplasia.

Mouse models of human ocular disease for translational research.

Mouse models provide a valuable tool for exploring pathogenic mechanisms underlying inherited human disease. Here, we describe seven mouse models identified through the Translational Vision Research Models (TVRM) program, each carrying a new allele of a gene previously linked to retinal developmental and/or degenerative disease. The mutations include four alleles of three genes linked to human nonsyndromic ocular diseases (Aipl1tvrm119, Aipl1tvrm127, Rpgrip1tvrm111, RhoTvrm334) and three alleles of genes associated with human syndromic diseases that exhibit ocular phentoypes (Alms1tvrm102, Clcn2nmf289, Fkrptvrm53). Phenotypic characterization of each model is provided in the context of existing literature, in some cases refining our current understanding of specific disease attributes. These murine models, on fixed genetic backgrounds, are available for distribution upon request and may be useful for understanding the function of the gene in the retina, the pathological mechanisms induced by its disruption, and for testing experimental approaches to treat the corresponding human ocular diseases.

Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease.

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.

Elevation of 20-carbon long chain bases due to a mutation in serine palmitoyltransferase small subunit b results in neurodegeneration.

Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Although sphingolipids with LCBs of other chain lengths have been identified, the functional significance of these low-abundance sphingolipids is unknown. The LCB chain length is determined by serine palmitoyltransferase (SPT) isoenzymes, which are trimeric proteins composed of two large subunits (SPTLC1 and SPTLC2 or SPTLC3) and a small subunit (SPTssa or SPTssb). Here we report the identification of an Sptssb mutation, Stellar (Stl), which increased the SPT affinity toward the C18 fatty acyl-CoA substrate by twofold and significantly elevated 20-carbon (C20) LCB production in the mutant mouse brain and eye, resulting in surprising neurodegenerative effects including aberrant membrane structures, accumulation of ubiquitinated proteins on membranes, and axon degeneration. Our work demonstrates that SPT small subunits play a major role in controlling SPT activity and substrate affinity, and in specifying sphingolipid LCB chain length in vivo. Moreover, our studies also suggest that excessive C20 LCBs or C20 LCB-containing sphingolipids impair protein homeostasis and neural functions.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.

Meckelin is necessary for photoreceptor intraciliary transport and outer segment morphogenesis.

PURPOSE: Cilia, complex structures found ubiquitously in most vertebrate cells, serve a variety of functions ranging from cell and fluid movement, cell signaling, tissue homeostasis, to sensory perception. Meckelin is a component of ciliary and cell membranes and is encoded by Tmem67 (Mks3). In this study, the retinal morphology and ciliary function in a mouse model for Meckel Syndrome Type 3 (MKS3) throughout the course of photoreceptor development was examined. METHODS: To study the effects of a disruption in the Mks3 gene on the retina, the authors introduced a functional allele of Pde6b into B6C3Fe a/a-bpck/J mice and evaluated their retinas by ophthalmoscopic, histologic, and ultrastructural examination. In addition, immunofluorescence microscopy was used to assess protein trafficking through the connecting cilium and to examine the localization of ciliary and synaptic proteins in Tmem67(bpck) mice and controls. RESULTS: Photoreceptors degenerate early and rapidly in bpck/bpck mutant mice. In addition, phototransduction proteins, such as rhodopsin, arrestin, and transducin, are mislocalized. Ultrastructural examination of photoreceptors reveal morphologically intact connecting cilia but dysmorphic and misoriented outer segment (OS) discs, at the earliest time point examined. CONCLUSIONS: These findings underscore the important role for meckelin in intraciliary transport of phototransduction molecules and their effects on subsequent OS morphogenesis and maintenance.

Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice.

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.

An ENU-induced mutation in the Mertk gene (Mertknmf12) leads to a slow form of retinal degeneration.

PURPOSE: To determine the basis and to characterize the phenotype of a chemically induced mutation in a mouse model of retinal degeneration. METHODS: Screening by indirect ophthalmoscopy identified a line of N-ethyl-N-nitrosourea (ENU) mutagenized mice demonstrating retinal patches. Longitudinal studies of retinal histologic sections showed photoreceptors in the peripheral retina undergoing slow, progressive degeneration. The mutation was named neuroscience mutagenesis facility 12 (nmf12), and mapping localized the critical region to Chromosome 2. RESULTS: Sequencing of nmf12 DNA revealed a point mutation in the c-mer tyrosine kinase gene, designated Mertk(nmf12). We detected elevated levels of tumor necrosis factor (Tnf, previously Tnfa) in retinas of Mertk(nmf12) homozygotes relative to wild-type controls and investigated whether the increase of TNF, an inflammatory cytokine produced by macrophages/monocytes that signals intracellularly to cause necrosis or apoptosis, could underlie the retinal degeneration observed in Mertk(nmf12) homozygotes. Mertk(nmf12) homozygous mice were mated to mice lacking the entire Tnf gene and partial coding sequences of the Lta (Tnfb) and Ltb (Tnfc) genes.(2) B6.129P2-Ltb/Tnf/Lta(tm1Dvk)/J homozygotes did not exhibit a retinal degeneration phenotype and will, hereafter, be referred to as Tnfabc(-/-) mice. Surprisingly, mice homozygous for both the Mertk(nmf12) and the Ltb/Tnf/Lta(tm1Dvk) allele (Tnfabc(-/-)) demonstrated an increase in the rate of retinal degeneration. CONCLUSIONS: These findings illustrate that a mutation in the Mertk gene leads to a significantly slower progressive retinal degeneration compared with other alleles of Mertk. These results demonstrate that TNF family members play a role in protecting photoreceptors of Mertk(nmf12) homozygotes from cell death.

NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development.

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

Mutations of the opsin gene (Y102H and I307N) lead to light-induced degeneration of photoreceptors and constitutive activation of phototransduction in mice.

Mutations in the Rhodopsin (Rho) gene can lead to autosomal dominant retinitis pigmentosa (RP) in humans. Transgenic mouse models with mutations in Rho have been developed to study the disease. However, it is difficult to know the source of the photoreceptor (PR) degeneration in these transgenic models because overexpression of wild type (WT) Rho alone can lead to PR degeneration. Here, we report two chemically mutagenized mouse models carrying point mutations in Rho (Tvrm1 with an Y102H mutation and Tvrm4 with an I307N mutation). Both mutants express normal levels of rhodopsin that localize to the PR outer segments and do not exhibit PR degeneration when raised in ambient mouse room lighting; however, severe PR degeneration is observed after short exposures to bright light. Both mutations also cause a delay in recovery following bleaching. This defect might be due to a slower rate of chromophore binding by the mutant opsins compared with the WT form, and an increased rate of transducin activation by the unbound mutant opsins, which leads to a constitutive activation of the phototransduction cascade as revealed by in vitro biochemical assays. The mutant-free opsins produced by the respective mutant Rho genes appear to be more toxic to PRs, as Tvrm1 and Tvrm4 mutants lacking the 11-cis chromophore degenerate faster than mice expressing WT opsin that also lack the chromophore. Because of their phenotypic similarity to humans with B1 Rho mutations, these mutants will be important tools in examining mechanisms underlying Rho-induced RP and for testing therapeutic strategies.

Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

Photoreceptor degeneration, azoospermia, leukoencephalopathy, and abnormal RPE cell function in mice expressing an early stop mutation in CLCN2.

PURPOSE: To determine the molecular basis and the pathologic consequences of a chemically induced mutation in a mouse model of photoreceptor degeneration, nmf240. METHODS: Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened by indirect ophthalmoscopy for abnormal fundi. A chromosomal position for the recessive nmf240 mutation was determined by a genome-wide linkage analysis by use of simple sequence length polymorphic markers in an F2 intercross. The critical region was refined, and candidate genes were screened by direct sequencing. The nmf240 phenotype was characterized by histologic analysis of the retina, brain, and male reproductive organs and by electroretinogram (ERG)-based studies of the retina and retinal pigment epithelium (RPE). RESULTS: Clinically, homozygous nmf240 mutants exhibit a grainy retina that progresses to panretinal patches of depigmentation. The mutation was localized to a region on chromosome 16 containing Clcn2, a gene associated with retinal degeneration. Sequencing identified a missense C-T mutation at nucleotide 1063 in Clcn2 that converts a glutamine to a stop codon. Mice homozygous for the Clcn2(nmf240) mutation experience a severe loss of photoreceptor cells at 14 days of age that is preceded by an elongation of RPE apical microvilli. Homozygous mutants also experience leukoencephalopathy in multiple brain areas and male sterility. Despite a normal retinal histology in nmf240 heterozygotes, the ERG light peak, generated by the RPE, is reduced. CONCLUSIONS: The nmf240 phenotype closely resembles that reported for Clcn2 knockout mice. The observation that heterozygous nmf240 mice present with a reduced ERG light peak component suggests that CLCN2 is necessary for the generation of this response component.

RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis.

The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1(tm1Tili) mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1(nmf247) that is phenotypically distinct from Rpgrip1(tm1Tili) mice. Photoreceptor degeneration in homozygous Rpgrip1(nmf247) mice is earlier in onset and more severe when compared with Rpgrip1(tm1Tili) mice. Also, ultrastructural studies reveal that whereas Rpgrip1(nmf247) mutants have a normal structure and number of connecting cilia, unlike Rpgrip1(tm1Tili) mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1(nmf247) mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.

Membrane frizzled-related protein is necessary for the normal development and maintenance of photoreceptor outer segments.

A 4 base pair deletion in a splice donor site of the Mfrp (membrane-type frizzled-related protein) gene, herein referred to as Mfrprd6/rd6, is predicted to lead to the skipping of exon 4 and photoreceptor degeneration in retinal degeneration 6 (rd6) mutant mice. Little, however, is known about the function of the protein or how the mutation causes the degenerative retinal phenotype. Here we examine ultrastructural changes in the retina of Mfrprd6/rd6 mice to determine the earliest effects of the mutation. We also extend the reported observations of the expression pattern of the dicistronic Mfrp/C1qtnf5 message and the localization of these and other retinal pigment epithelium (RPE) and retinal proteins during development and assess the ability of RPE cells to phagocytize outer segments (OSs) in mutant and wild-type (WT) mice. At the ultrastructural level, OSs do not develop normally in Mfrprd6/rd6 mutants. They are disorganized and become progressively shorter as mutant mice age. Additionally, there are focal areas in which there is a reduction of apical RPE microvilli. At P25, the rod electroretinogram (ERG) a-wave of Mfrprd6/rd6 mice is reduced in amplitude by ~50% as are ERG components generated by the RPE. Examination of beta-catenin localization and Fos and Tcf-1 expression, intermediates of the canonical Wnt pathway, showed that they were not different between mutant and WT mice, suggesting that MFRP may operate through an alternative pathway. Finally, impaired OS phagocytosis was observed in Mfrprd6/rd6 mice both in standard ambient lighting conditions and with bright light exposure when compared to WT controls.

Allelic variance between GRM6 mutants, Grm6nob3 and Grm6nob4 results in differences in retinal ganglion cell visual responses.

An electroretinogram (ERG) screen identified a mouse with a normal a-wave but lacking a b-wave, and as such it was designated no b-wave3 (nob3). The nob3 phenotype mapped to chromosome 11 in a region containing the metabotropic glutamate receptor 6 gene (Grm6). Sequence analyses of cDNA identified a splicing error in Grm6, introducing an insertion and an early stop codon into the mRNA of affected mice (designated Grm6(nob3)). Immunohistochemistry of the Grm6(nob3) retina showed that GRM6 was absent. The ERG and visual behaviour abnormalities of Grm6(nob3) mice are similar to Grm6(nob4) animals, and similar deficits were seen in compound heterozygotes (Grm6(nob4/nob3)), indicating that Grm6(nob3) is allelic to Grm6(nob4). Visual responses of Grm6(nob3) retinal ganglion cells (RGCs) to light onset were abnormal. Grm6(nob3) ON RGCs were rarely recorded, but when they were, had ill-defined receptive field (RF) centres and delayed onset latencies. When Grm6(nob3) OFF-centre RGC responses were evoked by full-field stimulation, significantly fewer converted that response to OFF/ON compared to Grm6(nob4) RGCs. Grm6(nob4/nob3) RGC responses verified the conclusion that the two mutants are allelic. We propose that Grm6(nob3) is a new model of human autosomal recessive congenital stationary night blindness. However, an allelic difference between Grm6(nob3) and Grm6(nob4) creates a disparity in inner retinal processing. Because the localization of GRM6 is limited to bipolar cells in the On pathway, the observed difference between RGCs in these mutants is likely to arise from differences in their inputs.