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Stem Cell Research & Therapy Dec 2023The CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases...
BACKGROUND
The CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function.
METHODS
We created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays.
RESULTS
We identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX cells in the early stage of retinal differentiation.
CONCLUSIONS
We confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies.
Topics: Animals; Humans; Mice; Cell Differentiation; Haploinsufficiency; Homeodomain Proteins; Organoids; Photoreceptor Cells; Retina; Trans-Activators
PubMed: 38049871
DOI: 10.1186/s13287-023-03590-3 -
Science Signaling Jul 2023Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the...
Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
Topics: Humans; Carrier Proteins; Cryoelectron Microscopy; Extracellular Matrix Proteins; Photoreceptor Cells; Synapses
PubMed: 37490546
DOI: 10.1126/scisignal.add9539 -
Cell Death & Disease Dec 2023Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events...
Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events has not been well delineated, primarily because of an inadequate understanding of the immunological processes involved in photoreceptor degeneration, especially that of peripheral leukocytes that infiltrate the subretinal space and retinal tissues. In this work, we characterized the role of leukocyte infiltration within the detached retina. We observed that CD45 CD11b Ly6G neutrophils and CD45 CD11b Ly6G Ly6C monocytes are the predominant peripheral immune cell populations that infiltrate the retinal and subretinal space after detachment. Selective depletion of monocytes or neutrophils using cell-specific targeting is neuroprotective for photoreceptors. These results indicate that peripheral innate immune cells contribute to photoreceptor degeneration, and targeting these immune cell populations could be therapeutic during retinal detachment.
Topics: Humans; Animals; Retinal Detachment; Monocytes; Neutrophils; Photoreceptor Cells; Retina; Retinal Degeneration; Photoreceptor Cells, Vertebrate; Disease Models, Animal
PubMed: 38102109
DOI: 10.1038/s41419-023-06350-6 -
Cellular and Molecular Life Sciences :... Nov 2023A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal...
A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult zebrafish retina of wild type AB strain (WT) and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. Retinas from both female and male fish were pooled to generate each library, combining data from both sexes. We provide a benchmark atlas of retinal cell type transcriptomes in zebrafish and insight into how each retinal cell type is affected in the P23H model. Oxidative stress is found throughout the retina, with increases in reliance on oxidative metabolism and glycolysis in the affected rods as well as cones, bipolar cells, and retinal ganglion cells. There is also transcriptional evidence for widespread synaptic remodeling and enhancement of glutamatergic transmission in the inner retina. Notably, changes in circadian rhythm regulation are detected in cones, bipolar cells, and retinal pigmented epithelium. We also identify the transcriptomic signatures of retinal progenitor cells and newly formed rods essential for the regenerative process. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.
Topics: Animals; Male; Female; Zebrafish; Retina; Retinitis Pigmentosa; Retinal Rod Photoreceptor Cells; Retinal Degeneration; Disease Models, Animal
PubMed: 37979052
DOI: 10.1007/s00018-023-05021-1 -
Ophthalmology Science Dec 2023Retinitis pigmentosa (RP) is the most common cause of inherited blindness, with onset occurring as early as 4 years of age in certain rare but severe forms caused by...
PURPOSE
Retinitis pigmentosa (RP) is the most common cause of inherited blindness, with onset occurring as early as 4 years of age in certain rare but severe forms caused by mutations in the gamma subunit of phosphodiesterase 6 (PDE6). Studies in humans and mice have shown that RP pathology begins with progressive photoreceptor death, which then drives changes in downstream neurons, neighboring retinal pigment epithelium (RPE), and vasculature. Here, we present the first detailed analysis of RP disease progression in -deficient mice.
DESIGN
Experimental study of an RP mouse model.
SUBJECTS
We studied and Pde6g mice at the age of 7, 16, 30, 44, and 56 days with n = 2 to 5 per group and time point.
METHODS
Photoreceptor degeneration and retinal remodeling were analyzed in retinal sections by immunofluorescence. Retinal blood vessel degradation was analyzed in flat-mounted retinas immunolabeled for isolectin GS-IB4. Protein expression was measured by immunoblot. Acellular capillaries were assessed in trypsin-digested and hematoxylin-eosin-stained retinas at postnatal day (P) 44. Retinal pigment epithelium cells were delineated in flat-mounted RPE-choroid-sclera by immunolabeling for the cell-adhesion protein β-catenin.
MAIN OUTCOME MEASURES
Immunofluorescence and morphometry (quantitative analysis of outer nuclear layer, dendrite area, vessel area, acellular vessels, RPE cell size, number of nuclei per RPE cell, RPE cell eccentricity, and RPE cell solidity).
RESULTS
This novel RP model exhibits early onset and rapid rod degeneration, with the vast majority gone by P16. This pathology leads to retinal remodeling, including changes of inner retinal neurons, early activation of glia cells, degradation of retinal vasculature, and structural abnormalities of the RPE.
CONCLUSIONS
The pathology in our mouse model precisely mirrors human RP progression. The results demonstrate the significant role of the gamma subunit in maintaining phosphodiesterase activity and provide new insights into the disease progression due to deficiency.
FINANCIAL DISCLOSURES
Proprietary or commercial disclosure may be found after the references.
PubMed: 37363133
DOI: 10.1016/j.xops.2023.100332 -
Cureus Oct 2023Retinitis Pigmentosa (RP) is an inherited retinal dystrophy (IRD) that causes progressive visual loss. Patients suffering from RP have a substantial influence on their... (Review)
Review
Retinitis Pigmentosa (RP) is an inherited retinal dystrophy (IRD) that causes progressive visual loss. Patients suffering from RP have a substantial influence on their everyday activities, social contacts, and jobs, lowering their quality of life. Frequent referral delays, as well as the lack of a standard therapy for the majority of patients, contribute to the significant unmet demand for RP. Any retinal injury has the potential to result in total blindness and visual impairment. Despite the fact that there is no cure for RP, people can manage it using rehabilitation programs and low-vision gadgets. The purpose of this research is to characterize the expanding treatment landscape for RP, as well as the justification for advanced therapy medicinal products (ATMPs). Vitamin A supplements can help prevent the sluggish visual loss caused by a prevalent kind of RP. The presence of visual purple in the rods and the underlying vascular choroid causes the retina to look purplish red. The major portion of the retina damaged is the rod photoreceptor electric cell; the development of diverse diseases is progressive. Because of the retina's accessibility, immunological privilege, and compartmentalization, hereditary retinal diseases are amenable to cell and gene therapy. Therapeutic techniques that attempt to rescue photoreceptors (gene therapies) require the existence of non-functional target cells, but other therapies (cell therapies) do not require the presence of live photoreceptors. To provide successful therapy choices for RP patients at all disease phases, the development pipeline must be continually diversified and advanced, as well as ongoing efforts to encourage early patient identification and quick diagnosis. Future research will focus on avoiding vision loss in genetic eye illnesses and assisting patients in regaining their eyesight. Retinal implants, cell therapies, supplementary medications, and gene therapies may become common treatments for reducing vision loss in the future.
PubMed: 38034182
DOI: 10.7759/cureus.48006 -
Proceedings of the National Academy of... Dec 2023Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons...
Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons for rod and cone signals en route to the brain, in both cases for the purpose of non-image vision as well as aspects of image vision. So far, six subtypes of ipRGCs (M1 through M6) have been characterized. Regarding their phototransduction mechanisms, we have previously found that, unconventionally, rhabdomeric (microvillous) and ciliary signaling motifs co-exist within a given M1-, M2-, and M4-ipRGC, with the first mechanism involving PLCβ4 and TRPC6,7 channels and the second involving cAMP and HCN channels. We have now examined M3-, M5-, and M6-cells and found that each cell likewise uses both signaling pathways for phototransduction, despite differences in the percentage representation by each pathway in a given ipRGC subtype for bright-flash responses (and saturated except for M6-cells). Generally, M3- and M5-cells show responses quite similar in kinetics to M2-responses, and M6-cell responses resemble broadly those of M1-cells although much lower in absolute sensitivity and amplitude. Therefore, similar to rod and cone subtypes in image vision, ipRGC subtypes possess the same phototransduction mechanism(s) even though they do not show microvilli or cilia morphologically.
Topics: Vision, Ocular; Light Signal Transduction; Retinal Ganglion Cells; Retinal Cone Photoreceptor Cells; Retinal Neurons; Rod Opsins
PubMed: 38109525
DOI: 10.1073/pnas.2315282120 -
Physiology (Bethesda, Md.) Jan 2024One of the biggest environmental alterations we have made to our species is the change in the exposure to light. During the day, we typically sit behind glass windows... (Review)
Review
One of the biggest environmental alterations we have made to our species is the change in the exposure to light. During the day, we typically sit behind glass windows illuminated by artificial light that is >400 times dimmer and has a very different spectrum than natural daylight. On the opposite end are the nights that are now lit up by several orders of magnitude. This review aims to provide food for thought as to why this matters for humans and other animals. Evidence from behavioral neuroscience, physiology, chronobiology, and molecular biology is increasingly converging on the conclusions that the biological nonvisual functions of light and photosensory molecules are highly complex. The initial work of von Frisch on extraocular photoreceptors in fish, the identification of rhodopsins as the molecular light receptors in animal eyes and eye-like structures and cryptochromes as light sensors in nonmammalian chronobiology, still allowed for the impression that light reception would be a relatively restricted, localized sense in most animals. However, light-sensitive processes and/or sensory proteins have now been localized to many different cell types and tissues. It might be necessary to consider nonlight-responding cells as the exception, rather than the rule.
Topics: Humans; Animals; Photoreceptor Cells, Invertebrate; Cryptochromes
PubMed: 37905983
DOI: 10.1152/physiol.00017.2023 -
The British Journal of Ophthalmology Nov 2023To describe and correlate electroretinographic responses with clinical and angiographic findings in retinal vasculitis (RV).
AIM
To describe and correlate electroretinographic responses with clinical and angiographic findings in retinal vasculitis (RV).
METHODS
Medical records of patients with diagnosis of RV at a tertiary eye centre from December 2017 to May 2021 were reviewed. Cases in which fluorescein angiography (FFA) and full field electroretinography (ffERG) were done within 1 month were included. FFAs were graded according to the Angiography Scoring for Uveitis Working Group from 0 to 40, where 0 is normal. A novel ffERG grading system was implemented where individual waves were graded for timing and amplitude and general ffERG score was determined with 6 being a perfect score.
RESULTS
20 patients (34 eyes) were included. Mean age was 43.9±19.8 years; 70% were female. Median best-corrected visual acuity was 0.8 (0.08-1). Mean FFA score was 12.6±6.5. Median general ffERG score was 5 (0-6). 68% and 91% of eyes had responses with general ffERG scores ≥5 and 4, respectively. Flicker timing was most commonly affected.FFA scores weakly correlated with delayed photopic cone b-wave and flicker timing (p=0.03 and 0.016, respectively). Vitreous haze moderately correlated with delayed cone b-wave timing (p<0.001), delayed flicker timing (p=0.002) and weakly correlated with lower flicker amplitude (p=0.03). Underlying systemic disease was associated with poor ffERG responses.
CONCLUSION
In this study, RV was not frequently associated with severe global retinal dysfunction Higher FFA scores, and vitreous haze grading were weakly, but significantly, correlated with cone-generated ffERG responses.
Topics: Humans; Female; Young Adult; Adult; Middle Aged; Male; Retina; Retinal Vasculitis; Electroretinography; Retinal Cone Photoreceptor Cells; Fluorescein Angiography
PubMed: 36130816
DOI: 10.1136/bjo-2022-321716 -
Cells Jun 2023Retinal detachment (RD) is a neurodegenerative blinding disease caused by plethora of clinical conditions. RD is characterized by the physical separation of retina from...
Retinal detachment (RD) is a neurodegenerative blinding disease caused by plethora of clinical conditions. RD is characterized by the physical separation of retina from the underlying retinal pigment epithelium (RPE), eventually leading to photoreceptor cell death, inflammation, and vision loss. Albeit the activation of complement plays a critical role in the pathogenesis of RD, the retinal cellular source for complement production remains elusive. Here, using C3 tdTomato reporter mice we show that retinal injury upregulates C3 expression, specifically in Müller cells. Activation of the complement cascade results in the generation of proinflammatory cleaved products, C3a and C5a, that bind C3aR and C5aR1, respectively. Our flow cytometry data show that retinal injury significantly upregulated C3aR and C5aR1 in microglia and resulted in the infiltration of peripheral immune cells. Loss of C3, C5, C3aR or C5aR1 reduced photoreceptor cell death and infiltration of microglia and peripheral immune cells into the sub-retinal space. These results indicate that C3/C3aR and C5/C5aR1 play a crucial role in eliciting photoreceptor degeneration and inflammatory responses in RD.
Topics: Mice; Animals; Ependymoglial Cells; Neuroinflammatory Diseases; Photoreceptor Cells; Cell Death; Retina; Retinal Detachment; Complement System Proteins
PubMed: 37443787
DOI: 10.3390/cells12131754