Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Bifidobacterium promotes retinal ganglion cell survival by regulating the balance of retinal glial cells.

INTRODUCTION: Optic nerve injury is a leading cause of irreversible blindness worldwide. The retinal ganglion cells (RGCs) and their axons cannot be regenerated once damaged. Therefore, reducing RGC damage is crucial to prevent blindness. Accordingly, we aimed to investigate the potential influence of the gut microbiota on RGC survival, as well as the associated action mechanisms. METHODS: We evaluated the effects of microbiota, specifically Bifidobacterium, on RGC. Optic nerve crush (ONC) was used as a model of optic nerve injury. Vancomycin and Bifidobacterium were orally administered to specific pathogen-free (SPF) mice. RESULTS: Bifidobacterium promoted RGC survival and optic nerve regeneration. The administration of Bifidobacterium inhibited microglia activation but promoted Müller cell activation, which was accompanied by the downregulation of inflammatory cytokines and upregulation of neurotrophic factors and retinal ERK/Fos signaling pathway activation. CONCLUSIONS: Our study demonstrates that Bifidobacterium-induced changes in intestinal flora promote RGC survival. The protective effect of Bifidobacterium on RGC can be attributed to the inhibition of microglia activation and promotion of Müller cell activation and the secondary regulation of inflammatory and neurotrophic factors.

Mechanism of Cone Degeneration in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of genetic disorders resulting in inherited blindness due to the degeneration of rod and cone photoreceptors. The various mechanisms underlying rod degeneration primarily rely on genetic mutations, leading to night blindness initially. Cones gradually degenerate after rods are almost eliminated, resulting in varying degrees of visual disability and blindness. The mechanism of cone degeneration remains unclear. An understanding of the mechanisms underlying cone degeneration in RP, a highly heterogeneous disease, is essential to develop novel treatments of RP. Herein, we review recent advancements in the five hypotheses of cone degeneration, including oxidative stress, trophic factors, metabolic stress, light damage, and inflammation activation. We also discuss the connection among these theories to provide a better understanding of secondary cone degeneration in RP. Five current mechanisms of cone degenerations in RP Interactions among different pathways are involved in RP.

Glycosaminoglycans Influence Extracellular Matrix of Human Trabecular Meshwork Cells Cultured on 3D Scaffolds.

Glaucoma is a multifactorial progressive optic neuropathy characterized by the loss of retinal ganglion cells leading to irreversible blindness. It is the leading cause of global irreversible blindness and is currently affecting over 70 million people. Elevated intraocular pressure (IOP) is considered the only modifiable risk factor and is a target of numerous treatment modalities. Researchers have assigned this elevation of IOP to accumulation of extracellular matrix (ECM) components in the aqueous humor (AH) outflow pathway. The major drainage structure for AH outflow is the trabecular meshwork (TM). The ECM of the TM is important in regulating IOP in both normal and glaucomatous eyes. In this work, we have studied the role of exogeneous glycosaminoglycans (GAGs), glucocorticoids, and culture conditions on the expression of the ECM gene and proteins by human TM (hTM) cells cultured on biomaterial scaffolds. Gene and protein expression levels of elastin, laminin, and matrix metalloproteinase-2 (MMP-2) were evaluated using quantitative PCR and immunohistochemistry. Pressure gradient changes in cell-laden scaffolds in perfusion cultures were also monitored. Our findings show that GAGs and dexamethasone play an influencing role in hTM ECM turnover at both transcriptional and translational levels by altering expression levels of elastin, laminin, and MMP-2. Understanding the role of exogeneous factors on hTM cell behavior is helpful in gaining insights on glaucoma pathogenesis and ultimately pivotal in development of novel therapeutics against the disease.

Corneal Epithelial Regeneration: Old and New Perspectives.

Corneal blindness is the fifth leading cause of blindness worldwide, and therapeutic options are still often limited to corneal transplantation. The corneal epithelium has a strong barrier function, and regeneration is highly dependent on limbal stem cell proliferation and basement membrane remodeling. As a result of the lack of corneal donor tissues, regenerative medicine for corneal diseases affecting the epithelium is an area with quite advanced basic and clinical research. Surgery still plays a prominent role in the treatment of epithelial diseases; indeed, innovative surgical techniques have been developed to transplant corneal and non-corneal stem cells onto diseased corneas for epithelial regeneration applications. The main goal of applying regenerative medicine to clinical practice is to restore function by providing viable cells based on the use of a novel therapeutic approach to generate biological substitutes and improve tissue functions. Interest in corneal epithelium rehabilitation medicine is rapidly growing, given the exposure of the corneal outer layers to external insults. Here, we performed a review of basic, clinical and surgical research reports on regenerative medicine for corneal epithelial disorders, classifying therapeutic approaches according to their macro- or microscopic target, i.e., into cellular or subcellular therapies, respectively.

Lifelong changes of neurotransmitter receptor expression and debilitation of hippocampal synaptic plasticity following early postnatal blindness.

In the weeks immediately after onset of sensory loss, extensive reorganization of both the cortex and hippocampus occurs. Two fundamental characteristics comprise widespread changes in the relative expression of GABA and glutamate receptors and debilitation of hippocampal synaptic plasticity. Here, we explored whether recovery from adaptive changes in the expression of plasticity-related neurotransmitter receptors and hippocampal synaptic plasticity occurs in the time-period of up to 12 months after onset of sensory loss. We compared receptor expression in CBA/J mice that develop hereditary blindness, with CBA/CaOlaHsd mice that have intact vision and no deficits in other sensory modalities throughout adulthood. GluN1-subunit expression was reduced and the GluN2A:GluN2B ratio was persistently altered in cortex and hippocampus. GABA-receptor expression was decreased and metabotropic glutamate receptor expression was altered. Hippocampal synaptic plasticity was persistently compromised in vivo. But although LTP in blind mice was chronically impaired throughout adulthood, a recovery of the early phase of LTP became apparent when the animals reached 12 months of age. These data show that cortical and hippocampal adaptation to early postnatal blindness progresses into advanced adulthood and is a process that compromises hippocampal function. A partial recovery of hippocampal synaptic plasticity emerges in advanced adulthood, however.

Vision-related convergent gene losses reveal SERPINE3's unknown role in the eye.

Despite decades of research, knowledge about the genes that are important for development and function of the mammalian eye and are involved in human eye disorders remains incomplete. During mammalian evolution, mammals that naturally exhibit poor vision or regressive eye phenotypes have independently lost many eye-related genes. This provides an opportunity to predict novel eye-related genes based on specific evolutionary gene loss signatures. Building on these observations, we performed a genome-wide screen across 49 mammals for functionally uncharacterized genes that are preferentially lost in species exhibiting lower visual acuity values. The screen uncovered several genes, including SERPINE3, a putative serine proteinase inhibitor. A detailed investigation of 381 additional mammals revealed that SERPINE3 is independently lost in 18 lineages that typically do not primarily rely on vision, predicting a vision-related function for this gene. To test this, we show that SERPINE3 has the highest expression in eyes of zebrafish and mouse. In the zebrafish retina, serpine3 is expressed in Müller glia cells, a cell type essential for survival and maintenance of the retina. A CRISPR-mediated knockout of serpine3 in zebrafish resulted in alterations in eye shape and defects in retinal layering. Furthermore, two human polymorphisms that are in linkage with SERPINE3 are associated with eye-related traits. Together, these results suggest that SERPINE3 has a role in vertebrate eyes. More generally, by integrating comparative genomics with experiments in model organisms, we show that screens for specific phenotype-associated gene signatures can predict functions of uncharacterized genes.

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.

Better Olfactory Performance and Larger Olfactory Bulbs in a Mouse Model of Congenital Blindness.

It is well established that early blindness results in enhancement of the remaining nonvisual sensory modalities accompanied by functional and anatomical brain plasticity. While auditory and tactile functions have been largely investigated, the results regarding olfactory functions remained less explored and less consistent. In the present study, we investigated olfactory function in blind mice using 3 tests: the buried food test, the olfactory threshold test, and the olfactory performance test. The results indicated better performance of blind mice in the buried food test and odor performance test while there was no difference in the olfactory threshold test. Using histological measurements, we also investigated if there was anatomical plasticity in the olfactory bulbs (OB), the most salient site for olfactory processing. The results indicated a larger volume of the OB driven by larger glomerular and granular layers in blind mice compared with sighted mice. Structural plasticity in the OB may underlie the enhanced olfactory performance in blind mice.

Blindness, Psychosis, and the Visual Construction of the World.

The relationship between visual loss and psychosis is complex: congenital visual loss appears to be protective against the development of a psychotic disorder, particularly schizophrenia. In later life, however, visual deprivation or visual loss can give rise to hallucinosis, disorders of visual insight such as blindsight or Anton syndrome, or, in the context of neurodegenerative disorders, more complex psychotic presentations. We draw on a computational psychiatric approach to consider the foundational role of vision in the construction of representations of the world and the effects of visual loss at different developmental stages. Using a Bayesian prediction error minimization model, we describe how congenital visual loss may be protective against the development of the kind of computational deficits postulated to underlie schizophrenia, by increasing the precision (and consequent stability) of higher-level (including supramodal) priors, focusing on visual loss-induced changes in NMDA receptor structure and function as a possible mechanistic substrate. In simple terms, we argue that when people cannot see from birth, they rely more heavily on the context they extract from the other senses, and the resulting model of the world is more impervious to the false inferences, made in the face of inevitably noisy perceptual input, that characterize schizophrenia. We show how a Bayesian prediction error minimization framework can also explain the relationship between later visual loss and other psychotic symptoms, as well as the effects of visual deprivation and hallucinogenic drugs, and outline experimentally testable hypotheses generated by this approach.

Bioactive Properties of Carotenoids in Human Health.

Research shows that certain bioactive compounds in our diet have beneficial effects on humanhealth[...].

The first steps in vision: cell types, circuits, and repair.

Dysfunction of the key sense of vision, leading to visual handicap or blindness, has a crucial effect on day-to-day life. In this commentary, I will summarize the work in my laboratory that is focused on a basic understanding of visual processing and the use of this information to understand disease mechanism and to develop correcting therapies. We are beginning to understand how cell types of the visual system interact in local circuits and compute visual information. This has brought insight into mechanisms of cell-type-specific diseases and has allowed us to design new therapies for restoring vision in genetic forms of blindness.

Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.

Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.

Impact of Global Mean Normalization on Regional Glucose Metabolism in the Human Brain.

Because the human brain consumes a disproportionate fraction of the resting body's energy, positron emission tomography (PET) measurements of absolute glucose metabolism (CMRglc) can serve as disease biomarkers. Global mean normalization (GMN) of PET data reveals disease-based differences from healthy individuals as fractional changes across regions relative to a global mean. To assess the impact of GMN applied to metabolic data, we compared CMRglc with and without GMN in healthy awake volunteers with eyes closed (i.e., control) against specific physiological/clinical states, including healthy/awake with eyes open, healthy/awake but congenitally blind, healthy/sedated with anesthetics, and patients with disorders of consciousness. Without GMN, global CMRglc alterations compared to control were detected in all conditions except in congenitally blind where regional CMRglc variations were detected in the visual cortex. However, GMN introduced regional and bidirectional CMRglc changes at smaller fractions of the quantitative delocalized changes. While global information was lost with GMN, the quantitative approach (i.e., a validated method for quantitative baseline metabolic activity without GMN) not only preserved global CMRglc alterations induced by opening eyes, sedation, and varying consciousness but also detected regional CMRglc variations in the congenitally blind. These results caution the use of GMN upon PET-measured CMRglc data in health and disease.

Eupatilin rescues ciliary transition zone defects to ameliorate ciliopathy-related phenotypes.

Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.

Genome-wide association meta-analysis highlights light-induced signaling as a driver for refractive error.

Refractive errors, including myopia, are the most frequent eye disorders worldwide and an increasingly common cause of blindness. This genome-wide association meta-analysis in 160,420 participants and replication in 95,505 participants increased the number of established independent signals from 37 to 161 and showed high genetic correlation between Europeans and Asians (>0.78). Expression experiments and comprehensive in silico analyses identified retinal cell physiology and light processing as prominent mechanisms, and also identified functional contributions to refractive-error development in all cell types of the neurosensory retina, retinal pigment epithelium, vascular endothelium and extracellular matrix. Newly identified genes implicate novel mechanisms such as rod-and-cone bipolar synaptic neurotransmission, anterior-segment morphology and angiogenesis. Thirty-one loci resided in or near regions transcribing small RNAs, thus suggesting a role for post-transcriptional regulation. Our results support the notion that refractive errors are caused by a light-dependent retina-to-sclera signaling cascade and delineate potential pathobiological molecular drivers.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Suppression of Melatonin Secretion in Totally Visually Blind People by Ocular Exposure to White Light: Clinical Characteristics.

PURPOSE: Although most totally visually blind individuals exhibit nonentrained circadian rhythms due to an inability of light to entrain the circadian pacemaker, a small proportion retain photic circadian entrainment, melatonin suppression, and other nonimage-forming responses to light. It is thought that these responses to light persist because of the survival of melanospin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), which project primarily to the circadian pacemaker and are functionally distinct from the rod and cone photoreceptors that mediate vision. We aimed to assess the integrity of nonimage-forming photoreception in totally visually blind patients with a range of ocular disorders. DESIGN: Within-subject, dark-controlled design. PARTICIPANTS: A total of 18 totally visually blind individuals (7 females; mean age ± standard deviation = 49.8±11.0 years) with various causes of blindness, including 3 bilaterally enucleated controls. METHODS: Melatonin concentrations were compared during exposure to a 6.5-hour bright white light (∼7000 lux) with melatonin concentrations measured 24 hours earlier at the corresponding clock times under dim-light (4 lux) conditions. MAIN OUTCOME MEASURES: Area under the curve (AUC) for melatonin concentration. RESULTS: Melatonin concentrations were significantly suppressed (defined as ≥33% suppression) during the bright-light condition compared with the dim-light condition in 5 of 15 participants with eyes (retinitis pigmentosa, n = 2; retinopathy of prematurity [ROP], n = 2; bilateral retinal detachments, n = 1). Melatonin concentrations remained unchanged in response to light in the remaining 10 participants with eyes (ROP, n = 3; optic neuritis/neuropathy, n = 2; retinopathy unknown, n = 2; congenital glaucoma, n = 1; congenital rubella syndrome, n = 1; measles retinopathy, n = 1) and in all 3 bilaterally enucleated participants. CONCLUSIONS: These data confirm that light-induced suppression of melatonin remains functionally intact in a minority of totally visually blind individuals with eyes. None of the bilaterally enucleated individuals or those with phthisis bulbi was responsive to light; of the remainder, half were responsive to light. Although inner retinal damage is associated with a high likelihood that nonimage-forming photoreception is absent, the impact of outer retinal damage is more ambiguous, and therefore the assessment of the presence, attenuation, or absence of nonimage-forming light responses in totally blind patients requires careful individual confirmation and cannot simply be assumed from the type of blindness.