CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Rod-derived cone viability factor promotes cone survival by stimulating aerobic glycolysis.

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.

Germline knockout of Nr2e3 protects photoreceptors in three distinct mouse models of retinal degeneration.

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targeting Nr2e3, a transcription factor required for the normal differentiation of rod photoreceptors. Nr2e3 knockout results in hybrid rod photoreceptors that express the full complement of rod genes, but also a subset of cone genes. We show that germline deletion of Nr2e3 potently protects rods in three mechanistically diverse mouse models of retinal degeneration caused by bright-light exposure (light damage), structural deficiency (rhodopsin-deficient Rho-/- mice), or abnormal phototransduction (phosphodiesterase-deficient rd10 mice). Nr2e3 knockout confers strong neuroprotective effects on rods without adverse effects on their gene expression, structure, or function. Furthermore, in all three degeneration models, prolongation of rod survival by Nr2e3 knockout leads to lasting preservation of cone morphology and function. These findings raise the possibility that upregulation of one or more cone genes in Nr2e3-deficient rods may be responsible for the neuroprotective effects we observe.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.

Chromophore supply modulates cone function and survival in retinitis pigmentosa mouse models.

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Retinal organoids from human-induced pluripotent stem cells: From studying retinal dystrophies to early diagnosis of Alzheimer's and Parkinson's disease.

Human-induced pluripotent stem cells (hiPSCs) have provided new methods to study neurodegenerative diseases. In addition to their wide application in neuronal disorders, hiPSCs technology can also encompass specific conditions, such as inherited retinal dystrophies. The possibility of evaluating alterations related to retinal disorders in 3D organoids increases the truthfulness of in vitro models. Moreover, both Alzheimer's (AD) and Parkinson's disease (PD) have been described as causing early retinal alterations, generating beta-amyloid protein accumulation, or affecting dopaminergic amacrine cells. This review addresses recent advances and future perspectives obtained from in vitro modeling of retinal diseases, focusing on retinitis pigmentosa (RP). Additionally, we depicted the possibility of evaluating changes related to AD and PD in retinal organoids obtained from potential patients long before the onset of the disease, constituting a valuable tool in early diagnosis. With this, we pointed out prospects in the study of retinal dystrophies and early diagnosis of AD and PD.

The Joubert-Meckel-Nephronophthisis Spectrum of Ciliopathies.

The Joubert syndrome (JS), Meckel syndrome (MKS), and nephronophthisis (NPH) ciliopathy spectrum could be the poster child for advances and challenges in Mendelian human genetics over the past half century. Progress in understanding these conditions illustrates many core concepts of human genetics. The JS phenotype alone is caused by pathogenic variants in more than 40 genes; remarkably, all of the associated proteins function in and around the primary cilium. Primary cilia are near-ubiquitous, microtubule-based organelles that play crucial roles in development and homeostasis. Protruding from the cell, these cellular antennae sense diverse signals and mediate Hedgehog and other critical signaling pathways. Ciliary dysfunction causes many human conditions termed ciliopathies, which range from multiple congenital malformations to adult-onset single-organ failure. Research on the genetics of the JS-MKS-NPH spectrum has spurred extensive functional work exploring the broadly important role of primary cilia in health and disease. This functional work promises to illuminate the mechanisms underlying JS-MKS-NPH in humans, identify therapeutic targets across genetic causes, and generate future precision treatments.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Combination of blockade of endothelin signalling and compensation of IGF1 expression protects the retina from degeneration.

Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, affecting 1 in 4000 people worldwide. This disease is currently assumed to be intractable, because effective therapeutic methods have not been established, regardless of genetic or sporadic traits. Here, we examined a RP mouse model in which the Prominin-1 (Prom1) gene was deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, conducted single-cell expression profiling, and compared the gene expression with and without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, Igf1 was decreased in rod photoreceptor cells and astrocytes under the light-stimulated condition. Consistently, the insulin-like growth factor (IGF) signal was weakened in light-stimulated photoreceptor cells. The recovery of Igf1 expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, thereby resulting in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in maintaining retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.

Disease-specific variant interpretation highlighted the genetic findings in 2325 Japanese patients with retinitis pigmentosa and allied diseases.

BACKGROUND: As gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa (RP)-related genes in a large number of Japanese patients with RP by applying the standardised variant interpretation guidelines for Japanese patients with IRD (J-IRD-VI guidelines) built upon the American College of Medical Genetics and Genomics and the Association for Molecular Pathology rules, and assess the contribution of these genes in RP-allied diseases. METHODS: We assessed 2325 probands with RP (n=2155, including n=1204 sequenced previously with the same sequencing panel) and allied diseases (n=170, newly analysed), including Usher syndrome, Leber congenital amaurosis and cone-rod dystrophy (CRD). Target sequencing using a panel of 86 genes was performed. The variants were interpreted according to the J-IRD-VI guidelines. RESULTS: A total of 3564 variants were detected, of which 524 variants were interpreted as pathogenic or likely pathogenic. Among these 524 variants, 280 (53.4%) had been either undetected or interpreted as variants of unknown significance or benign variants in our earlier study of 1204 patients with RP. This led to a genetic diagnostic rate in 38.6% of patients with RP, with EYS accounting for 46.7% of the genetically solved patients, showing a 9% increase in diagnostic rate from our earlier study. The genetic diagnostic rate for patients with CRD was 28.2%, with RP-related genes significantly contributing over other allied diseases. CONCLUSION: A large-scale genetic analysis using the J-IRD-VI guidelines highlighted the population-specific genetic findings for Japanese patients with IRD; these findings serve as a foundation for the clinical application of gene-specific therapies.

Tsc2 knockout counteracts ubiquitin-proteasome system insufficiency and delays photoreceptor loss in retinitis pigmentosa.

SignificanceStudies in multiple experimental systems have demonstrated that an increase in proteolytic capacity of post-mitotic cells improves cellular resistance to a variety of stressors, delays cellular aging and senescence. Therefore, approaches to increase the ability of cells to degrade misfolded proteins could potentially be applied to the treatment of a broad spectrum of human disorders. An example would be retinal degenerations, which cause irreversible loss of vision and are linked to impaired protein degradation. This study suggests that chronic activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway in degenerating photoreceptor neurons could stimulate the degradation of ubiquitinated proteins and enhance proteasomal activity through phosphorylation.

Preclinical dose response study shows NR2E3 can attenuate retinal degeneration in the retinitis pigmentosa mouse model RhoP23H+/.

Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over 280 genes which ultimately result in blindness. Modifier genes play a key role in modulating disease phenotypes, and mutations in them can affect disease outcomes, rate of progression, and severity. Our previous studies have demonstrated that the nuclear hormone receptor 2 family e, member 3 (Nr2e3) gene reduced disease progression and loss of photoreceptor cell layers in RhoP23H-/- mice. This follow up, pharmacology study evaluates a longitudinal NR2E3 dose response in the clinically relevant heterozygous RhoP23H mouse. Reduced retinal degeneration and improved retinal morphology was observed 6 months following treatment evaluating three different NR2E3 doses. Histological and immunohistochemical analysis revealed regions of photoreceptor rescue in the treated retinas of RhoP23H+/- mice. Functional assessment by electroretinogram (ERG) showed attenuated photoreceptor degeneration with all doses. This study demonstrates the effectiveness of different doses of NR2E3 at reducing retinal degeneration and informs dose selection for clinical trials of RhoP23H-associated RP.

Atypical retinopathy in ataxia with vitamin E deficiency: report of a sibship.

Typical retinitis pigmentosa (RP) may not be the only retinal phenotype encountered in ataxia with vitamin E deficiency (AVED). The following short case series describes a novel form of retinopathy in AVED. We describe two patients with AVED belonging to the same consanguineous sibship. Both presented an unusual retinopathy consisting of scattered, multifocal, nummular, hyperautofluorescent atrophic retinal patches. The retinopathy remained stable under vitamin E supplementation. We hypothesize these changes to be the result of arrested AVED-related RP following early supplementation with α-tocopherol acetate.

Navigating the future of retinitis pigmentosa treatments: A comprehensive analysis of therapeutic approaches in rd10 mice.

Retinitis pigmentosa (RP) is a degenerative disease, caused by genetic mutations that lead to a loss in photoreceptors. For research on RP, rd10 mice, which carry mutations in the phosphodiesterase (PDE) gene, exhibit degenerative patterns comparable to those of patients with RP, making them an ideal model for investigating potential treatments. Although numerous studies have reported the potential of biochemical drugs, gene correction, and stem cell transplantation in decelerating rd10 retinal degeneration, a comprehensive review of these studies has yet to be conducted. Therefore, here, a comparative analysis of rd10 mouse treatment research over the past decade was performed. Our findings suggest that biochemical drugs capable of inhibiting the inflammatory response may be promising therapeutics. Additionally, significant progress has been made in the field of gene therapy; nevertheless, challenges such as strict delivery requirements, bystander editing, and off-target effects still need to be resolved. Nevertheless, secretory function is the only unequivocal protective effect of stem cell transplantation. In summary, this review presents a comprehensive analysis and synthesis of the treatment approaches employing rd10 mice as experimental subjects, describing a clear pathway for future RP treatment research and identifies potential clinical interventions.

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion.

Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Chromenone derivatives as novel pharmacological chaperones for retinitis pigmentosa-linked rod opsin mutants.

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with a natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilize rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused by either bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.