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.

To evaluate short-term markers of outcome in diabetic macular edema (DME).

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2A R) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2A R blockade directly in microglia. Here we show that blocking microglial A2A R prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure-induced death. The A2A R antagonist SCH 58261 or the knockdown of A2A R expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2A R antagonist decreased microglia proliferation, as well as the expression and release of pro-inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2A R blockade recapitulated the effects of microglia depletion, suggesting that blocking A2A R in microglia is able to control neurodegeneration in glaucoma-like conditions. Importantly, in human organotypic retinal cultures, A2A R blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2A R as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

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.

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.

A top priority in biomarker development for Alzheimer's disease (AD) and Parkinson's disease (PD) is the focus on early diagnosis, where the use of the retina is a promising avenue of research. We computed fundus images from optical coherence tomography (OCT) data and analysed the structural arrangement of the retinal tissue using texture metrics. We built clinical class classification models to distinguish between healthy controls (HC), AD, and PD, using machine learning (support vector machines). Median sensitivity is 88.7%, 79.5% and 77.8%, for HC, AD, and PD eyes, respectively. When the same subject has the same classification for both eyes, 94.4% (median) of the classifications are correct. A significant amount of information discriminating between multiple neurodegenerative states is conveyed by OCT imaging of the human retina, even when differences in thickness are not yet present. This technique may allow for simultaneously diagnosing Alzheimer's and Parkinson's diseases.

Glaucoma is a degenerative disease that causes irreversible loss of vision and is characterized by retinal ganglion cell (RGC) loss. Others and we have demonstrated that chronic neuroinflammation mediated by reactive microglial cells plays a role in glaucomatous pathology. Exosomes are extracellular vesicles released by most cells, including microglia, that mediate intercellular communication. The role of microglial exosomes in glaucomatous degeneration remains unknown. Taking the prominent role of microglial exosomes in brain neurodegenerative diseases, we studied the contribution of microglial-derived exosomes to the inflammatory response in experimental glaucoma. Microglial cells were exposed to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure, the main risk factor for glaucoma. Naïve microglia (BV-2 cells or retinal microglia) were exposed to exosomes derived from BV-2 cells under EHP conditions (BV-Exo-EHP) or cultured in control pressure (BV-Exo-Control). We found that BV-Exo-EHP increased the production of pro-inflammatory cytokines, promoted retinal microglia motility, phagocytic efficiency, and proliferation. Furthermore, the incubation of primary retinal neural cell cultures with BV-Exo-EHP increased cell death and the production of reactive oxygen species. Exosomes derived from retinal microglia (MG-Exo-Control or MG-Exo-EHP) were injected in the vitreous of C57BL/6J mice. MG-Exo-EHP sustained activation of retinal microglia, mediated cell death, and impacted RGC number. Herein, we show that exosomes derived from retinal microglia have an autocrine function and propagate the inflammatory signal in conditions of elevated pressure, contributing to retinal degeneration in glaucomatous conditions.

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.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness, characterized by optic nerve damage and retinal ganglion cell (RGC) death. Elevated intraocular pressure (IOP) is a main risk factor of glaucoma. Neuroinflammation plays an important role in glaucoma. We have been demonstrating that elevated pressure triggers microglia reactivity that contribute to the loss of RGCs. Adenosine, acting on adenosine receptors, is a crucial modulator of microglia phenotype. Microglia express all adenosine receptors. Previously, we demonstrated that the activation of adenosine A3 receptor (A3R) affords protection to the retina, including RGCs, unveiling the possibility for a new strategy for glaucoma treatment. Since microglial cells express A3R, we now studied the ability of a selective A3R agonist (2-Cl-IB-MECA) in controlling microglia reactivity induced by elevated hydrostatic pressure (EHP), used to mimic elevated IOP. The activation of A3R reduced EHP-induced inducible nitric oxide synthase (iNOS) expression, microglia migration and phagocytosis in BV-2 cells. In retinal microglia, proliferation and phagocytosis elicited by EHP were also decreased by A3R activation. This work demonstrates that 2-Cl-IB-MECA, the selective agonist of A3R, is able to hinder microglia reactivity, suggesting that A3R agonists could afford protection against glaucomatous degeneration through the control of neuroinflammation.

Mice are widely used as models for many diseases, including eye and neurodegenerative diseases. However, there is a lack of normative data for retinal thickness over time, especially at young ages. In this work, we present a normative thickness database from one to four-months-old, for nine layers/layer-aggregates, including the total retinal thickness, obtained from the segmentation of spectral-domain optical coherence tomography (SD-OCT) data from the C57BL6/129S mouse strain. Based on fifty-seven mice, this normative database provides an opportunity to study the ageing of control mice and characterise disease models' ageing, such as the triple transgenic mouse model of Alzheimer's disease (3×Tg-AD) used in this work. We report thickness measurements, the differences in thickness per layer, demonstrate a nasal-temporal asymmetry, and the variation of thickness as a function to the distance to the optic disc centre. Significant differences were found between the transgenic group's thickness and the normative database for the entire period covered in this study. Even though it is well accepted that retinal nerve fibre layer (RNFL) thinning is a hallmark of neurodegeneration, our results show a thicker RNFL-GCL (RNFL-Ganglion cell layer) aggregate for the 3×Tg-AD mice until four-months-old.

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.

This study aims to investigate the relationship between structural changes in the retina and white matter in the brain, in early Alzheimer's disease (AD). Twenty-three healthy controls (mean age = 63.4±7.5 years) and seventeen AD patients (mean age = 66.5±6.6 years) were recruited for this study. By combining two imaging techniques-optical coherence tomography and diffusion tensor imaging (DTI)-the association between changes in the thickness of individual retinal layers and white matter dysfunction in early AD was assessed. Retinal layers were segmented, and thickness measurements were obtained for each layer. DTI images were analyzed with a quantitative data-driven approach to evaluating whole-brain diffusion metrics, using tract-based spatial statistics. Diffusion metrics, such as fractional anisotropy, are markers for white matter integrity. Multivariate and partial correlation analyses evaluating the association between individual retinal layers thickness and diffusion metrics were performed. We found that axial diffusivity, indexing axonal integrity, was significantly reduced in AD (p = 0.016, Cohen's d = 1.004) while in the retina, only a marginal trend for significance was found for the outer plexiform layer (p = 0.084, Cohen's d = 0.688). Furthermore, a positive association was found in the AD group between fractional anisotropy and the inner nuclear layer thickness (p < 0.05, r = 0.419, corrected for multiple comparisons by controlling family-wise error rate). Our findings suggest that axonal damage in the brain dominates early on in this condition and shows an association with retinal structural integrity already at initial stages of AD. These findings are consistent with an early axonal degeneration mechanism in AD.

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.

It has been claimed that the retina can be used as a window to study brain disorders. However, concerning Alzheimer's disease (AD), it still remains controversial whether changes occurring in the brain and retina are associated. We aim to understand when changes start appearing in the retina and brain, how changes progress, and if they are correlated.

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.