Epithelial mechanics are maintained by inhibiting cell fusion with age in Drosophila.

A characteristic of normal aging and age-related diseases is the remodeling of the cellular organization of a tissue through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that abdominal epithelium of the adult fruit fly becomes polyploid with age through generation of multinucleated cells by cell fusion. Inhibition of fusion does not improve the lifespan of the fly, but does enhance its biomechanical fitness, a measure of the healthspan of the animal. Remarkably, Drosophila can maintain their epithelial tension and abdominal movements with age when cell fusion is inhibited. Epithelial cell fusion also appears to be dependent on a mechanical cue, as knockdown of Rho kinase, E-cadherin or α-catenin is sufficient to induce multinucleation in young animals. Interestingly, mutations in α-catenin in mice result in retina pigment epithelial multinucleation associated with macular disease. Therefore, we have discovered that polyploid cells arise by cell fusion and contribute to the decline in the biomechanical fitness of the animal with age.

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.

Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the global aging population. Familial aggregation and genome-wide association (GWA) studies have identified gene variants associated with AMD, implying a strong genetic contribution to AMD development. Two loci, on human Chr 1q31 and 10q26, respectively, represent the most influential of all genetic factors. While the role of CFH at Chr 1q31 is well established, uncertainty remains about the genes ARMS2 and HTRA1, at the Chr 10q26 locus. Since both genes are in strong linkage disequilibrium, assigning individual gene effects is difficult. In this chapter, we review current literature about ARMS2 and HTRA1 and their relevance to AMD risk. Future studies will be necessary to unravel the mechanisms by which they contribute to AMD.

Single-Cell RNA Sequencing Reveals Molecular Features of Heterogeneity in the Murine Retinal Pigment Epithelium.

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity within native tissue, which adds complexity to global transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2667 and 2846 RPE cells. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell populations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited the hallmarks of stem and/or progenitor (SP) cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and SP gene expression and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means clustering of all detected transcripts identified additional expression patterns that may advance the understanding of RPE SP cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue.

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.

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' 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.

A Dpagt1 Missense Variant Causes Degenerative Retinopathy without Myasthenic Syndrome in Mice.

Congenital disorders of glycosylation (CDG) are a heterogenous group of primarily autosomal recessive mendelian diseases caused by disruptions in the synthesis of lipid-linked oligosaccharides and their transfer to proteins. CDGs usually affect multiple organ systems and vary in presentation, even within families. There is currently no cure, and treatment is aimed at ameliorating symptoms and improving quality of life. Here, we describe a chemically induced mouse mutant, tvrm76, with early-onset photoreceptor degeneration. The recessive mutation was mapped to Chromosome 9 and associated with a missense mutation in the Dpagt1 gene encoding UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase (EC 2.7.8.15). The mutation is predicted to cause a substitution of aspartic acid with glycine at residue 166 of DPAGT1. This represents the first viable animal model of a Dpagt1 mutation and a novel phenotype for a CDG. The increased expression of Ddit3, and elevated levels of HSPA5 (BiP) suggest the presence of early-onset endoplasmic reticulum (ER) stress. These changes were associated with the induction of photoreceptor apoptosis in tvrm76 retinas. Mutations in human DPAGT1 cause myasthenic syndrome-13 and severe forms of a congenital disorder of glycosylation Type Ij. In contrast, Dpagt1tvrm76 homozygous mice present with congenital photoreceptor degeneration without overt muscle or muscular junction involvement. Our results suggest the possibility of DPAGT1 mutations in human patients that present primarily with retinitis pigmentosa, with little or no muscle disease. Variants in DPAGT1 should be considered when evaluating cases of non-syndromic retinal degeneration.

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.

The Impact of Adherens and Tight Junctions on Physiological Function and Pathological Changes in the Retina.

The formation of solid tissues is not a simple aggregation of individual cells but rather an ordered assembly of cells connected by junctions. These junctions provide a diffusion barrier as well as mechanical support and a conduit for signalling changes in the environment to the cells. Cell junctions are functionally categorized as occluding (e.g. tight junctions, TJs), anchoring (e.g. adherens junctions, AJs) and communicating junctions (e.g. gap junctions). Each type of the cell junction is formed by protein complexes with extracellular domains and/or intracellular domains, which bind partners that provide scaffolding and signalling components. Cell junctions are ubiquitously expressed in multiple tissues and organs, including the retina. In the retina, their biological impact is not limited to regulating tissue growth and development. Disruption of the complexes mediates both congenital and postnatal pathogenesis. In this review, we will focus on cell junctions, specifically AJs and TJs in the external limiting membrane, in order to articulate their influence on pathophysiology of the retina.

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.