To retrospectively analyze the clinical characteristics of patients who were screened for mutations with the ATP-binding cassette transporter gene ABCA4 (ABCA4) microarray in a routine clinical DNA diagnostics setting.

Butterfly-shaped pigment dystrophy is an eye disease characterized by lesions in the macula that can resemble the wings of a butterfly. Here we report the identification of heterozygous missense mutations in the CTNNA1 gene (encoding α-catenin 1) in three families with butterfly-shaped pigment dystrophy. In addition, we identified a Ctnna1 missense mutation in a chemically induced mouse mutant, tvrm5. Parallel clinical phenotypes were observed in the retinal pigment epithelium (RPE) of individuals with butterfly-shaped pigment dystrophy and in tvrm5 mice, including pigmentary abnormalities, focal thickening and elevated lesions, and decreased light-activated responses. Morphological studies in tvrm5 mice demonstrated increased cell shedding and the presence of large multinucleated RPE cells, suggesting defects in intercellular adhesion and cytokinesis. This study identifies CTNNA1 gene variants as a cause of macular dystrophy, indicates that CTNNA1 is involved in maintaining RPE integrity and suggests that other components that participate in intercellular adhesion may be implicated in macular disease.

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

The formation of a functionally integrated nervous system is dependent on a highly organized sequence of events that includes timely division and differentiation of progenitors. Several apical polarity proteins have been shown to play crucial roles during neurogenesis, however, the role of Crumbs 2 (CRB2) in cortical development has not previously been reported. Here, we show that conditional ablation of Crb2 in the murine dorsal telencephalon leads to defects in the maintenance of the apical complex. Furthermore, within the mutant dorsal telencephalon there is premature expression of differentiation proteins. We examined the physiological function of Crb2 on wild type genetic background as well as on background lacking Crb1. Telencephalon lacking CRB2 resulted in reduced levels of PALS1 and CRB3 from the apical complex, an increased number of mitotic cells and expanded neuronal domain. These defects are transient and therefore only result in rather mild cortical abnormalities. We show that CRB2 is required for maintenance of the apical polarity complex during development of the cortex and regulation of cell division, and that loss of CRB2 results in cortical abnormalities.

Chronic central serous chorioretinopathy (cCSC) is an eye disease characterized by an accumulation of serous fluid under the retina. It is postulated that this fluid accumulation results from hyperpermeability and swelling of the choroid, the underlying vascular tissue of the eye, causing a dysfunction of the retinal pigment epithelium. This fluid accumulation causes neuroretinal detachment. A prolonged neuroretinal detachment in the macula can lead to permanent vision loss. Therefore, treatment is aimed primarily at achieving resolution of subretinal fluid, preferably within the first 4 months after diagnosis of the disease. A broad spectrum of treatment modalities has been investigated in cCSC, but no consensus exists on the optimal treatment of cCSC. Currently, photodynamic therapy (PDT) and high-density subthreshold micropulse laser treatment (HSML) are among the most frequently cited treatments in obtaining successful neuroretinal reattachment.

Recently, treatment with MEK inhibitors has been shown to be an effective treatment option for metastatic melanoma. Treatment efficacy is dependent on inhibition of MAPK-related melanoma proliferation. However, targeting of MEK can be accompanied by a time-dependent and reversible serous retinopathy of unknown origin.We analyzed the molecular mechanism by which the MEK inhibitor binimetinib may lead to retinopathy, using neuroretina and cell models of retinal pigment epithelium (RPE).Binimetinib inhibited the MAPK pathway while discontinuation of treatment resulted in reactivation. However, cell proliferation was not inhibited correspondingly during binimetinib treatment of ARPE19 cells. Remarkably, post-mitotic neuroretinal tissue displayed a strong MAPK activation that was lost after binimetinib treatment.We propose that binimetinib-associated retinopathy is correlated with inhibition of the MAPK pathway in multiple retinal components. Retinal cells are able to regain the activation after binimetinib treatment, mimicking the reversibility of the retinopathy. As most retinal cells are nonregenerating, other mechanisms than stimulation of proliferation must be involved.

Mutations in the PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin-protein ligase (PARKIN) genes are associated with familial forms of Parkinson's disease (PD). PINK1, a protein kinase, and PARKIN, an E3 ubiquitin ligase, control the specific elimination of dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial network and preserving energy metabolism. PINK1 regulates PARKIN translocation in impaired mitochondria and drives their removal via selective autophagy, a process known as mitophagy. As knowledge obtained using different PINK1 and PARKIN transgenic animal models is being gathered, growing evidence supports the contribution of mitophagy impairment to several human pathologies, including PD and Alzheimer's diseases (AD). Therefore, therapeutic interventions aiming to modulate PINK1/PARKIN signalling might have the potential to treat these diseases. In this review, we will start by discussing how the interplay of PINK1 and PARKIN signalling helps mediate mitochondrial physiology. We will continue by debating the role of mitochondrial dysfunction in disorders such as amyotrophic lateral sclerosis, Alzheimer's, Huntington's and Parkinson's diseases, as well as eye diseases such as age-related macular degeneration and glaucoma, and the causative factors leading to PINK1/PARKIN-mediated neurodegeneration and neuroinflammation. Finally, we will discuss PINK1/PARKIN gene augmentation possibilities with a particular focus on AD, PD and glaucoma.

The recently emerged pachychoroid concept has changed the understanding of age-related macular degeneration (AMD), which is a major cause of blindness; recent studies attributed AMD in part to pachychoroid disease central serous chorioretinopathy (CSC), suggesting the importance of elucidating the CSC pathogenesis. Our large genome-wide association study followed by validation studies in three independent Japanese and European cohorts, consisting of 1546 CSC samples and 13,029 controls, identified two novel CSC susceptibility loci: TNFRSF10A-LOC389641 and near GATA5 (rs13278062, odds ratio = 1.35, P = 1.26 × 10-13; rs6061548, odds ratio = 1.63, P = 5.36 × 10-15). A T allele at TNFRSF10A-LOC389641 rs13278062, a risk allele for CSC, is known to be a risk allele for AMD. This study not only identified new susceptibility genes for CSC, but also improves the understanding of the pathogenesis of AMD.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM+ cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1 KO Crb2 LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1 KO Crb2 LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1 KO Crb2 LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1 KO Crb2 LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Iron accumulation causes cell death and disrupts tissue functions, which necessitates chelation therapy to reduce iron overload. However, clinical utilization of deferoxamine (DFO), an iron chelator, has been documented to give rise to systemic adverse effects, including ocular toxicity. This study provided the pathogenic and molecular basis for DFO-related retinopathy and identified retinal pigment epithelium (RPE) as the target tissue in DFO-related retinopathy. Our modeling demonstrated the susceptibility of RPE to DFO compared with the neuroretina. Intriguingly, we established upregulation of hypoxia inducible factor (HIF) 2α and mitochondrial deficit as the most prominent pathogenesis underlying the RPE atrophy. Moreover, suppressing hyperactivity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) protects the RPE against lesions both in vitro and in vivo. This supported our observation that AKG supplementation alleviates visual impairment in a patient undergoing DFO-chelation therapy. Overall, our study established a significant role of iron deficiency in initiating DFO-related RPE atrophy. Inhibiting HIF2α and rescuing mitochondrial function by AKG protect RPE cells and can potentially ameliorate patients' visual function.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.

The transforming growth factor-β (TGF-β) family is known to play critical roles in cancer progression. While the dual role of TGF-β is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo.

This study investigated the phenotypic spectrum of PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and early-onset cataract) syndrome caused by biallelic variants in the ABHD12 gene. A total of 15 patients from 12 different families were included, with a mean age of 36.7 years (standard deviation [SD] ± 11.0; range from 17.5 to 53.9) at the most recent examination. The presence and onset of neurological, audiological and ophthalmic symptoms were variable, with no evident order of symptom appearance. The mean best-corrected visual acuity was 1.1 logMAR (SD ± 0.9; range from 0.1 to 2.8; equivalent to 20/250 Snellen) and showed a trend of progressive decline. Different types of cataract were observed in 13 out of 15 patients (87%), which also included congenital forms of cataract. Fundus examination revealed macular involvement in all patients, ranging from alterations of the retinal pigment epithelium to macular atrophy. Intraretinal spicular hyperpigmentation was observed in 7 out of 15 patients (47%). From an ophthalmic perspective, clinical manifestations in patients with PHARC demonstrate variability with regard to their onset and severity. Given the variable nature of PHARC, an early multidisciplinary assessment is recommended to assess disease severity.

To determine whether extremely mild small vessel disease (SVD) phenotypes can occur in NOTCH3 variant carriers from Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) pedigrees using clinical, genetic, neuroimaging, and skin biopsy findings.

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.