Disruption in murine Eml1 perturbs retinal lamination during early development.

During mammalian development, establishing functional neural networks in stratified tissues of the mammalian central nervous system depends upon the proper migration and positioning of neurons, a process known as lamination. In particular, the pseudostratified neuroepithelia of the retina and cerebrocortical ventricular zones provide a platform for progenitor cell proliferation and migration. Lamination defects in these tissues lead to mispositioned neurons, disrupted neuronal connections, and abnormal function. The molecular mechanisms necessary for proper lamination in these tissues are incompletely understood. Here, we identified a nonsense mutation in the Eml1 gene in a novel murine model, tvrm360, displaying subcortical heterotopia, hydrocephalus and disorganization of retinal architecture. In the retina, Eml1 disruption caused abnormal positioning of photoreceptor cell nuclei early in development. Upon maturation, these ectopic photoreceptors possessed cilia and formed synapses but failed to produce robust outer segments, implying a late defect in photoreceptor differentiation secondary to mislocalization. In addition, abnormal positioning of Müller cell bodies and bipolar cells was evident throughout the inner neuroblastic layer. Basal displacement of mitotic nuclei in the retinal neuroepithelium was observed in tvrm360 mice at postnatal day 0. The abnormal positioning of retinal progenitor cells at birth and ectopic presence of photoreceptors and secondary neurons upon maturation suggest that EML1 functions early in eye development and is crucial for proper retinal lamination during cellular proliferation and development

Deficiency in Lyst function leads to accumulation of secreted proteases and reduced retinal adhesion.

Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology

Genetic Interaction between Mfrp and Adipor1 Mutations Affect Retinal Disease Phenotypes

Adipor1tm1Dgen and Mfrprd6 mutant mice share similar eye disease characteristics. Previously, studies established a functional relationship of ADIPOR1 and MFRP proteins in maintaining retinal lipidome homeostasis and visual function. However, the independent and/or interactive contribution of both genes to similar disease phenotypes, including fundus spots, decreased axial length, and photoreceptor degeneration has yet to be examined. We performed a gene-interaction study where homozygous Adipor1tm1Dgen and Mfrprd6 mice were bred together and the resulting doubly heterozygous F1 offspring were intercrossed to produce 210 F2 progeny. Four-month-old mice from all nine genotypic combinations obtained in the F2 generation were assessed for white spots by fundus photo documentation, for axial length by caliper measurements, and for photoreceptor degeneration by histology. Two-way factorial ANOVA was performed to study individual as well as gene interaction effects on each phenotype. Here, we report the first observation of reduced axial length in Adipor1tmlDgen homozygotes. We show that while Adipor1 and Mfrp interact to affect spotting and degeneration, they act independently to control axial length, highlighting the complex functional association between these two genes. Further examination of the molecular basis of this interaction may help in uncovering mechanisms by which these genes perturb ocular homeostasis

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina. Mol Ther 2018 Feb 7 26(2):524-541

A Splicing Mutation in Slc4a5 Results in Retinal Detachment and Retinal Pigment Epithelium Dysfunction

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5\u27 splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model.

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans

Lysosomal Trafficking Regulator (LYST).

Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. Adv Exp Med Biol 2016; 854:745-50

Molecular characterization of TUB, TULP1, and TULP2, members of the novel tubby gene family and their possible relation to ocular diseases

Tubby, an autosomal recessive mutation, mapping to mouse chromosome 7, was recently found to be the result of a splicing defect in a novel gene with unknown function. Database searches revealed that sequences corresponding to the C terminus of the tub protein were highly conserved across a number of species including humans, mice, Caenorhabditis elegans, Arabidopsis, rice, and maize, and that tub was a member of a gene family. We describe here, TUB, the human homolog of mouse tub, and two newly characterized family members, TULP1 for tubby like protein 1 and TULP2. These three family members, which differ in the N-terminal half of the protein, share 60–90% amino acid identity across their conserved C-terminal region and have distinct tissue expression patterns. Alternatively spliced transcripts with 5′ variable sequences, three of which have been identified for the tubby gene, may mediate tissue specific expression. We also report that TUB, TULP1, and TULP2 map to human chromosomes 11p15.4, 6p21.3, and 19q13.1, respectively. TULP1 and TULP2 map within the minimal intervals identified for retinitis pigmentosa 14 on chromosome 6p21.3 and cone-rod dystrophy on chromosome 19q13.1. TULP1 and TULP2, which are expressed in the retina, make excellent candidates for these ocular diseases as a mutation within the tub gene is known to lead to early progressive retinal degeneration

Clinical utility gene card for: Alström Syndrome - update 2013.

Eur J Hum Genet 2013 Nov; 21(11)