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Choroidal neovascularization (CNV) is a pathological process in which aberrant blood vessels invade the subretinal space of the mammalian eye. It is a characteristic feature of the prevalent neovascular age-related macular degeneration (nAMD). Circulating microRNAs (cmiRNAs) are regarded as potentially valuable biomarkers for various age-related diseases, including nAMD. Here, we investigated cmiRNA expression in an established laser-induced CNV mouse model. Upon CNV induction in C57Bl/6 mice, blood-derived cmiRNAs were initially determined globally by RNA next generation sequencing, and the most strongly dysregulated cmiRNAs were independently replicated by quantitative reverse transcription PCR (RT-qPCR) in blood, retinal, and retinal pigment epithelium (RPE)/choroidal tissue. Our findings suggest that two miRNAs, mmu-mir-486a-5p and mmur-mir-92a-3p, are consistently dysregulated during CNV formation. Furthermore, in functional in vitro assays, a significant impact of mmu-mir-486a-5p and mmu-mir-92a-3p on murine microglial cell viability was observed, while mmu-mir-92a-3p also showed an impact on microglial mobility. Taken together, we report a robust dysregulation of two miRNAs in blood and RPE/choroid after laser-induced initiation of CNV lesions in mice, highlighting their potential role in pathology and eventual therapy of CNV-associated complications.

Purpose: Avascular tissues can be used to identify antilymph- or antihemangiogenic factors. The human sclera-the outer covering layer of the eye, lacks lymphatic vessels and contains only a superficial network of blood vessels and was used here to identify endogenous antiangiogenic factors. Methods: Expression levels of a panel of 96 known pro- and antiangiogenic factors were analyzed in 12 scleral or conjunctival control samples from normal human donors using real-time PCR. In vitro, scleral homogenate was cocultured with blood- and lymphatic endothelial cells (BECs and LECs) and immunohistochemistry was performed of scleral fibroblasts and BECs. Results: Three antiangiogenic factors were significantly upregulated in the human sclera compared to the conjunctiva, including FBLN5 (fibulin 5), SERPINF1 (serpin peptidase inhibitor, clade F, member 1 = pigment epithelium derived factor) and TIMP2 (Tissue inhibitor of metalloproteinases 2). Six proangiogenic factors were significantly downregulated in the sclera, including FLT4 (Fms-related tyrosine kinase 4=VEGF-R3), HGF (hepatocyte growth factor), KIT (CD117 / c-kit), PROX1 (prospero homeobox 1), SEMA3F (semaphorin-3F) and TGFA (transforming growth factor alpha). In vitro, scleral homogenate inhibited the growth of both BECs and LECs. Immunohistochemistry labeling of three major antiangiogenic factors from scleral tissue confirmed TIMP3 and PEDF expression both in scleral fibroblasts and in blood endothelial cells, whereas TIMP2 was not detectable. Conclusion: Balancing anti- and proangiogenic factors actively regulates human scleral avascularity, inhibits endothelial cell growth in vitro, and thus may help maintaining the vascular privilege of the inner eye.

Purpose: Part of the hidden genetic variation in heterogeneous genetic conditions such as inherited retinal diseases (IRDs) can be explained by copy-number variations (CNVs). Here, we explored the genomic landscape of IRD genes listed in RetNet to identify and prioritize those genes susceptible to CNV formation. Methods: RetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants and literature. CNVs identified in an IRD cohort were characterized using targeted locus amplification (TLA) on extracted genomic DNA. Results: Exhaustive literature mining revealed 1,345 reported CNVs in 81 different IRD genes. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence of IRD genes. Moreover, we identified and delineated 30 new CNVs in IRD cases, 13 of which are novel and three of which affect noncoding, putative cis-regulatory regions. Finally, the breakpoints of six complex CNVs were determined using TLA in a hypothesis-neutral manner. Conclusion: We propose a ranking of CNV-prone IRD genes and demonstrate the efficacy of TLA for the characterization of CNVs on extracted DNA. Finally, this IRD-oriented CNV study can serve as a paradigm for other genetically heterogeneous Mendelian diseases with hidden genetic variation.

Mitochondrial Translocator protein (18 kDa) (TSPO) is strongly expressed in reactive microglia and serves as a therapeutic target for alleviation of neuronal degeneration. However, little is known about TSPO's transcriptional regulation in microglia. The aim of this study was to identify genetic elements and transcription factors required for basal and inducible TSPO expression in microglia. Murine Tspo promoter was cloned into the pGL4.10 luciferase vector and functionally characterized in BV-2 cells. Deletion mutagenesis indicated that 845 bases upstream were sufficient to reconstitute near maximal promoter activity in BV-2. Deletion of 593 to 520 sequences, which harbour an Apl, Ets.2 and Nkx3.1 site which also serves as a non-canonical binding site for Spl-family transcription factors, led to a dramatic decrease in both basal and LPS induced promoter activity. Further deletion of 168 to 39 sequences, which contains four GC boxes, also led to a significant decrease in promoter activity. Targeted mutations of Apl, Ets.2, Nkx3.1/Spl/3/4 and the GC boxes led to significant decreases in promoter activity. ChIP-qPCR revealed that Pu.1, Apl, Stat3, Spl, Sp3 and Sp4 bind to the endogenous Tspo promoter. Notably, binding of these factors, with the exception of Stat3, was significantly enhanced upon LPS treatment. RNAi silencing of Pu.1, cJun, cFos, Spl, Sp3, Sp4 and Stat3 strongly lowered Tspo promoter activity while Apl silencing inhibited LPS induced increase in Tspo protein levels. These findings demonstrate that consensus binding sequences for Apl, Ets.2, distal as well as proximal Sp1/3/4 sites regulate basal and LPS induced Tspo promoter activity in microglia.

Mutations in the Norrin (NDP) gene cause severe developmental blood vessel defects in the retina leading to congenital blindness. In the retina of Ndph-knockout mice only the superficial capillary network develops. Here, a detailed characterization of this mouse model at late stages of the disease using in vivo retinal imaging revealed cystoid structures that closely resemble the ovoid cysts in the inner nuclear layer of the human retina with cystoid macular edema (CME). In human CME an involvement of Müller glia cells is hypothesized. In Ndph-knockout retinae we could demonstrate that activated Müller cells were located around and within these cystoid spaces. In addition, we observed extensive activation of retinal microglia and development of neovascularization. Furthermore, ex vivo analyses detected extravasation of monocytic cells suggesting a breakdown of the blood retina barrier. Thus, we could demonstrate that also in the developmental retinal vascular pathology present in the Ndph-knockout mouse inflammatory processes are active and may contribute to further retinal degeneration. This observation delivers a new perspective for curative treatments of retinal vasculopathies. Modulation of inflammatory responses might reduce the symptoms and improve visual acuity in these diseases.

Background: Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear. Methods: Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis. Results: Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924). Conclusions: ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch's membrane, ARMS2 protein defi

Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the un-coated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration. (C) 2017 Elsevier B.V. All rights reserved.

A chronic activation of the congenital immune system that is detrimental to the course of the disease underlies all forms of hereditary retinal degeneration investigated in detail so far. Above all, resident microglial cells of the retina and various proteins of the soluble complement system contribute to damage to photoreceptors and the retinal pigment epithelium. Specific target structures on reactive immune cells in addition to dysregulated soluble immune regulators offer a new starting point for treatments to promote the survival of the retina, despite a genetic predisposition to degeneration. In this article, we provide an insight into the essential regulatory mechanisms of retinal immunology, discuss the potential use of immunological biomarkers for diagnosing retinal conditions, and demonstrate immunomodulatory therapies through the use of biological agents and endogenous messengers.

The aim of this study was to unravel the molecular pathogenesis of an unusual retinitis pigmentosa (RP) phenotype observed in a Turkish consanguineous family. Homozygosity mapping revealed two candidate genes, SAMD7 and RHO. A homozygous RHO mutation c.448G > A, p.E150K was found in two affected siblings, while no coding SAMD7 mutations were identified. Interestingly, four non-coding homozygous variants were found in two SAMD7 genomic regions relevant for binding of the retinal transcription factor CRX (CRX-bound regions, CBRs) in these affected siblings. Three variants are located in a promoter CBR termed CBR1, while the fourth is located more downstream in CBR2. Transcriptional activity of these variants was assessed by luciferase assays and electroporation of mouse retinal explants with reporter constructs of wild-type and variant SAMD7 CBRs. The combined CBR2/CBR1 variant construct showed significantly decreased SAMD7 reporter activity compared to the wild-type sequence, suggesting a cis-regulatory effect on SAMD7 expression. As Samd7 is a recently identified Crx-regulated transcriptional repressor in retina, we hypothesize that these SAMD7 variants might contribute to the retinal phenotype observed here, characterized by unusual, recognizable pigment deposits, differing from the classic spicular intraretinal pigmentation observed in other individuals homozygous for p. E150K, and typically associated with RP in general.

Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals' lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.

The aim of this study was to unravel the molecular pathogenesis of an unusual retinitis pigmentosa (RP) phenotype observed in a Turkish consanguineous family. Homozygosity mapping revealed two candidate genes, SAMD7 and RHO. A homozygous RHO mutation c.448G > A, p.E150K was found in two affected siblings, while no coding SAMD7 mutations were identified. Interestingly, four non-coding homozygous variants were found in two SAMD7 genomic regions relevant for binding of the retinal transcription factor CRX (CRX-bound regions, CBRs) in these affected siblings. Three variants are located in a promoter CBR termed CBR1, while the fourth is located more downstream in CBR2. Transcriptional activity of these variants was assessed by luciferase assays and electroporation of mouse retinal explants with reporter constructs of wild-type and variant SAMD7 CBRs. The combined CBR2/CBR1 variant construct showed significantly decreased SAMD7 reporter activity compared to the wild-type sequence, suggesting a cis-regulatory effect on SAMD7 expression. As Samd7 is a recently identified Crx-regulated transcriptional repressor in retina, we hypothesize that these SAMD7 variants might contribute to the retinal phenotype observed here, characterized by unusual, recognizable pigment deposits, differing from the classic spicular intraretinal pigmentation observed in other individuals homozygous for p.E150K, and typically associated with RP in general., SCOPUS: ar.j, info:eu-repo/semantics/published

Resident microglial cells can be regarded as the immunological watchdogs of the brain and the retina. They are active sensors of their neuronal microenvironment and rapidly respond to various insults with a morphological and functional transformation into reactive phagocytes. There is strong evidence from animal models and in situ analyses of human tissue that microglial reactivity is a common hallmark of various retinal degenerative and inflammatory diseases. These include rare hereditary retinopathies such as retinitis pigmentosa and X-linked juvenile retinoschisis but also comprise more common multifactorial retinal diseases such as age-related macular degeneration, diabetic retinopathy, glaucoma, and uveitis as well as neurological disorders with ocular manifestation. In this review, we describe how microglial function is kept in balance under normal conditions by cross-talk with other retinal cells and summarize how microglia respond to different forms of retinal injury. In addition, we present the concept that microglia play a key role in local regulation of complement in the retina and specify aspects of microglial aging relevant for chronic inflammatory processes in the retina. We conclude that this resident immune cell of the retina cannot be simply regarded as bystander of disease but may instead be a potential therapeutic target to be modulated in the treatment of degenerative and inflammatory diseases of the retina. (C) 2014 Elsevier Ltd. All rights reserved.

Purpose: The aim of this study was to identify the genetic cause of early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy in a consanguineous family. Methods: An affected 6-month-old child underwent neurological and ophthalmological examinations. Genetic analyses included homozygosity mapping, copy number analysis, conventional polymerase chain reaction, Sanger sequencing, quantitative polymerase chain reaction, and whole-exome sequencing. Expression analysis of GRID2 was performed by quantitative polymerase chain reaction and immunohistochemistry. Results: A homozygous deletion of exon 2 of GRID2 (p.G1y30_Glu-81del) was identified in the proband. GRID2 encodes an ionotropic glutamate receptor known to be selectively expressed in cerebellar Purkinje cells. Here, we demonstrated GRID2 expression in human adult retina and retinal pigment epithelium. In addition, Grid2 expression was demonstrated in different stages of murine retinal development. GRID2 immunostaining was shown in murine and human retina. Whole-exome sequencing in the proband did not provide arguments for other disease-causing mutations, supporting the idea that the phenotype observed represents a single clinical entity. Conclusion: We identified GRID2 as an underlying disease gene of early-onset autosomal recessive cerebellar ataxia with retinal dystrophy, expanding the clinical spectrum of GRID2 deletion mutants. We demonstrated for the first time GRID2 expression and localization in human and murine retina, providing evidence for a novel functional role of GRID2 in the retina. Genet Med advance online publication 14 August 2014

Mutations in the acid sphingomyelinase (aSMase) coding gene sphingomyelin phosphodiesterase 1 (SMPD1) cause Niemann-Pick disease (NPD) type A and B. Sphingomyelin storage in cells of the mononuclear phagocyte system cause hepatosplenomegaly and severe neurodegeneration in the brain of NPD patients. However, the effects of aSMase deficiency on retinal structure and microglial behavior have not been addressed in detail yet. Here, we demonstrate that retinas of aSMase(-/-) mice did not display overt neuronal degeneration but showed significantly reduced scotopic and photopic responses in electroretinography. In vivo fundus imaging of aSMase(-/-) mice showed many hyperreflective spots and staining for the retinal microglia marker Iba1 revealed massive proliferation of retinal microglia that had significantly enlarged somata. Nile red staining detected prominent phospholipid inclusions in microglia and lipid analysis showed significantly increased sphingomyelin levels in retinas of aSMase(-/-) mice. In conclusion, the aSMase-deficient mouse is the first example in which microglial lipid inclusions are directly related to a loss of retinal function. (C) 2015 Elsevier Inc. All rights reserved.

In the healthy retina, microglial cells represent a self-renewing population of innate immune cells, which constantly survey their microenvironment. Equipped with receptors, a microglial cell detects subtle cellular damage and rapidly responds with activation, migration, and increased phagocytic activity. While the involvement of microglial cells has been well characterized in monogenic retinal disorders, it is still unclear how they contribute to the onset of retinal aging disorders including age-related macular degeneration (AMD). There is evidence, that microglial activation is not solely a secondary manifestation of retinal tissue damage in age-related disorders. Thus, work in the aging rodent and human retina suggests that long-lived and genetically predisposed microglia transform into a dystrophic state, with loss of neuroprotective functions. In this concept, malfunction of aging microglia can trigger a chronic low-grade inflammatory environment that favors the onset and progression of retinal degeneration.

Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive disorder that leads to progressive retinal degeneration with a distinct electroretinogram (ERG) phenotype. CDSRR patients show reduced sensitivity to dim light, augmented response to suprathreshold light and reduced response to flicker. The disorder is caused by mutations in the KCNV2 gene, which encodes the Kv11.1 subunit of a voltage-gated potassium channel. Here, we studied the retina-specific expression and cis-regulatory activity of the murine Kcnv2 gene using electroporation of explanted retinas. Using qRT-PCR profiling of early postnatal retinas, we showed that Kcnv2 expression increased towards P14, which marks the beginning of visual activity in mice. In vivo electroporation of GFP-Kcnv2 expressing plasmids revealed that Kv11.1 localizes to the inner segment membranes of adult P21 photoreceptors. Using bioinformatic prediction and chromatin immuno-precipitation (ChIP), we identified two Crx binding sites (CBS) and one Nrl binding site (NBS) in the Kcnv2 promoter. Reporter electroporation of the wild type promoter region induced strong DsRed expression, indicating high regulatory activity, whereas shRNA-mediated knockdown of Crx and Nrl resulted in reduced Kcnv2 promoter activity and low endogenous Kcnv2 mRNA expression in the retina. Site-directed mutagenesis of the CBS and NBS demonstrated that CBS2 is crucial for Kcnv2 promoter activity. We conclude that nucleotide changes in evolutionary conserved CBS could impact retina-specific expression levels of Kcnv2.

Background: The translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions. Methods: TSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173. Results: TSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their ne

PURPOSE. We studied associations of genetic polymorphisms in age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) in nonagenarians with age-related macular degeneration (AMD). METHODS. This case-control study comprised 2737 persons (1204 controls, 1433 AMD cases), including 166 nonagenarians (52 controls, 114 AMD cases). Single nucleotide polymorphisms (SNPs) in the genes ARMS2 and CFH were determined. Risk scores were computed by multiple logistic regression analysis, including genetic and environmental risk factors (smoking, hypertension, body mass index, diabetes) for different age groups (<70, 70-79, 80-89, >= 90 years [nonagenarians]). RESULTS. In nonagenarians, ARMS2 showed the weakest associations with AMD (odds ratio [OR] = 1.52, P = 0.127) compared to the other groups (OR, 70 years = 2.23, P = 1.03 x 10(-13); OR, 70-79 years = 2.70, P = 1.00 x 10(-13); OR, 80-89 years = 3.11, P = 6.56 x 10(-8)). For CFH, ORs for AMD increased with age (<70 years OR = 1.96, P = 1.80 x 10(-11); 70-79 years OR = 1.89, P = 4.48 x 10(-13); 80-89 years OR = 2.71, P = 1.28 x 10(-7)), but decreased again in the nonagenarians (OR = 2.21, P = 0.005). Compared to the group <70 years, reduced minor allele frequencies (MAFs) for AMD patients were observed in the nonagenarians (CFH 0.54 vs. 0.43, P = 0.009; ARMS2 0.44 vs. 0.29, P = 2.97 x 10(-5)), while the MAFs in controls were not significantly different. The genetic risk score revealed the lowest discriminative power in the nonagenarians with an area-under-curve (AUC) of 0.658 for receiver-operating characteristics (AUC 80-89 years = 0.768, 70-79 years = 0.704, <70 years = 0.682), while no significant difference was seen for the environmental risk score (AUC <70 years = 0.579, 70-79 years = 0.567, 80-89 years = 0.600, >90 years = 0.608). CONCLUSIONS. Risk alleles in CFH and ARMS2 have a significantly smaller effect on AMD development in nonagenarians, while environmental factors retain a similar effect.