In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1 −/− ) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1 −/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex-that is, instead of a single ubiquitous channel, VRACs could be formed by cell type-or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.bestrophin 1 | volume-regulated anion channel | induced pluripotent stem cell | retinal pigment epithelium | mouse sperm

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-014-8308-8) contains supplementary material, which is available to authorized users.

Mutations in bestrophin-1 (BEST1) are associated with distinct retinopathies, notably three forms with autosomal dominant inheritance and one condition with an autosomal recessive mode of transmission. The molecular mechanisms underlying their distinct retinal phenotypes are mostly unknown. Although heterozygous missense mutations in BEST1 reveal dominant-negative effects in patients with autosomal dominant Best disease (BD), heterozygous mutations associated with autosomal recessive bestrophinopathy (ARB) display no disease phenotype. Here we show that the recessive mutations trigger a strong and fast protein degradation process in the endoplasmic reticulum (ER), thereby favoring a decreased stoichiometry of mutant versus normal BEST1 subunits in the assembly of the homo-pentameric BEST1 chloride channel. In contrast, dominant mutations escape ER-associated degradation and are subjected to a slightly delayed post-ER degradation via the endo-lysosomal degradation pathway. As a result, increased formation of a non-functional BEST1 channel occurs due to a roughly equimolar incorporation of normal and mutant BEST1 subunits into the channel complex. Taken together, our data provide insight into the molecular pathways of dominantly and recessively acting BEST1 missense mutations suggesting that the site of subcellular protein quality control as well as the rate and degree of mutant protein degradation are ultimately responsible for the distinct retinal disease phenotypes in BD and ARB.

Best vitelliform macular dystrophy (BD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and the autosomal recessive bestrophinopathy (ARB), together known as the bestrophinopathies, are caused by mutations in the bestrophin-1 (BEST1) gene affecting anion transport through the plasma membrane of the retinal pigment epithelium (RPE). To date, while no treatment exists a better understanding of BEST1-related pathogenesis may help to define therapeutic targets. Here, we systematically characterize functional consequences of mutant BEST1 in thirteen RPE patient cell lines differentiated from human induced pluripotent stem cells (hiPSCs). Both BD and ARB hiPSC-RPEs display a strong reduction of BEST1-mediated anion transport function compared to control, while ADVIRC mutations trigger an increased anion permeability suggesting a stabilized open state condition of channel gating. Furthermore, BD and ARB hiPSC-RPEs differ by the degree of mutant protein turnover and by the site of subcellular protein quality control with adverse effects on lysosomal pH only in the BD-related cell lines. The latter finding is consistent with an altered processing of catalytic enzymes in the lysosomes. The present study provides a deeper insight into distinct molecular mechanisms of the three bestrophinopathies facilitating functional categorization of the more than 300 known BEST1 mutations that result into the distinct retinal phenotypes.

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined PADJ = 1.15 × 10−6, OR 1.332 (1.187–1.496)]. This association was characterized by a highly significant sex difference (Pdiff = 0.0032) in that it was clearly confined to females with genome-wide significance [PADJ = 2.62 × 10−8, OR 1.541 (1.324–1.796); males: PADJ = 0.382, OR 1.084 (0.905–1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMD pathogenesis. Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-015-8342-1) contains supplementary material, which is available to authorized users.

Aim:We aimed to compare the quantity and quality of aging retinal organoids generated by applying three distinct differentiation protocols for human-derived induced pluripotent stem cells (hiPSC).Methods: hiPSC were differentiated to retinal organoids using a 3D technique (Method 1) and a 3D-2D-3D technique (Method 2), the latter modified by the addition of BMP4 (Method 3). To investigate the retinal organoid quantity, we counted the number of retinal domains, precursors to organoids during differentiation. The retinal organoid quality was evaluated by immunostaining for markers of different retinal cell types in whole cryosections after days 85, 120, and 200 in culture.Results: Method 3 produced strikingly more retinal domains per differentiation (65 ± 27) than Methods 1 (12.3 ± 11.2) and 2 (6.3 ± 6.7). Furthermore, retinal organoids differentiated with Method 3 contained significantly more CRX-positive photoreceptors and BRN3A-positive ganglion cells after 85 days in culture, compared to Methods 1 and 2. After 200 days in culture, the retinal organoids differentiated with Method 3 showed proper maturation, as demonstrated by the expression of mature rod and cone photoreceptor markers. Conclusion:This study demonstrates that the retinal organoid differentiation method can significantly impact the cellular composition of retinal organoids at various time points of development.

Human bestrophin-1 (BEST1) is an integral membrane protein known to function as a Ca 2+ -activated and volume-regulated chloride channel. The majority of disease-associated mutations in BEST1 constitute missense mutations and were shown in vitro to lead to a reduction in mutant protein half-life causing Best disease (BD), a rare autosomal dominant macular dystrophy. To further delineate BEST1-associated pathology in vivo and to provide an animal model useful to explore experimental treatment efficacies, we have generated a knock-in mouse line (Best1 Y227N ). Heterozygous and homozygous mutants revealed no significant ocular abnormalities up to 2 years of age. In contrast, knock-in animals demonstrated a severe phenotype in the male reproductive tract. In heterozygous Best1 Y227N males, Best1 protein was significantly reduced in testis and almost absent in homozygous mutant mice, although mRNA transcription of wild-type and knock-in allele is present and similar in quantity. Degradation of mutant Best1 protein in testis was associated with adverse effects on sperm motility and the capability to fertilize eggs. Based on these results, we conclude that mice carrying the Best1 Y227N mutation reveal a reproducible pathologic phenotype and thus provide a valuable in vivo tool to evaluate efficacy of drug therapies aimed at restoring Best1 protein stability and function.

ZusammenfassungDas Bestrophin-1-Gen (BEST1) codiert für ein integrales Membranprotein und findet sich im basolateralen Aspekt des retinalen Pigmentepithels. Mutationen im BEST1 sind mit einer heterogenen Gruppe von erblichen Netzhautdystrophien assoziiert, den sog. Bestrophinopathien, die schon in jüngerem Alter zu einer Beeinträchtigung des Sehvermögens führen können. Die einzelnen Krankheitsbilder sind anhand ihrer phänotypischen Merkmale und Erbgänge abgrenzbar. Während die meisten BEST1-Mutationen einem autosomal-dominanten Erbgang folgen, bei dem bereits eine defekte Genkopie zur Erkrankung führt, sind bei der autosomal-rezessiven Bestrophinopathie heterozygote Mutationsträger i. d. R. symptomfrei. In diesem Übersichtsartikel soll am Beispiel der Bestrophinopathien die Bedeutung der Kenntnis von mutationsabhängigen Mechanismen für das Verständnis der Pathogenese, aber auch für die Entwicklung von zukünftigen Therapieansätzen, hervorgehoben werden.