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Cell & Bioscience May 2024Retinitis pigmentosa (RP) is a genetically heterogeneous group of degenerative disorders causing progressive vision loss due to photoreceptor death. RP affects other...
BACKGROUND
Retinitis pigmentosa (RP) is a genetically heterogeneous group of degenerative disorders causing progressive vision loss due to photoreceptor death. RP affects other retinal cells, including the retinal pigment epithelium (RPE). MicroRNAs (miRs) are implicated in RP pathogenesis, and downregulating miR-181a/b has shown therapeutic benefit in RP mouse models by improving mitochondrial function. This study investigates the expression profile of miR-181a/b in RPE cells and the neural retina during RP disease progression. We also evaluate how miR-181a/b downregulation, by knocking out miR-181a/b-1 cluster in RPE cells, confers therapeutic efficacy in an RP mouse model and explore the mechanisms underlying this process.
RESULTS
Our findings reveal distinct expression profiles, with downregulated miR-181a/b in RPE cells suggesting a protective response and upregulated miR-181a/b in the neural retina indicating a role in disease progression. We found that miR-181a/b-2, encoded in a separate genomic cluster, compensates for miR-181a/b-1 ablation in RPE cells at late time points. The transient downregulation of miR-181a/b in RPE cells at post-natal week 6 (PW6) led to improved RPE morphology, retarded photoreceptor degeneration and decreased RPE aerobic glycolysis.
CONCLUSIONS
Our study elucidates the underlying mechanisms associated with the therapeutic modulation of miR-181a/b, providing insights into the metabolic processes linked to its RPE-specific downregulation. Our data further highlights the impact of compensatory regulation between miR clusters with implications for the development of miR-based therapeutics.
PubMed: 38773556
DOI: 10.1186/s13578-024-01243-3 -
Nature Communications May 2024As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape....
As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.
Topics: Animals; Actins; Mice; Retinitis Pigmentosa; Eye Proteins; Cilia; Humans; Retinal Photoreceptor Cell Outer Segment; Mice, Knockout; Mice, Inbred C57BL; Cell Membrane
PubMed: 38773095
DOI: 10.1038/s41467-024-48639-w -
PloS One 2024Zeaxanthin dipalmitate (ZD) is a chemical extracted from wolfberry that protects degenerated photoreceptors in mouse retina. However, the pure ZD is expensive and hard...
Zeaxanthin dipalmitate (ZD) is a chemical extracted from wolfberry that protects degenerated photoreceptors in mouse retina. However, the pure ZD is expensive and hard to produce. In this study, we developed a method to enrich ZD from wolfberry on a production line and examined whether it may also protect the degenerated mouse retina. The ZD-enriched wolfberry extract (ZDE) was extracted from wolfberry by organic solvent method, and the concentration of ZD was identified by HPLC. The adult C57BL/6 mice were treated with ZDE or solvent by daily gavage for 2 weeks, at the end of the first week the animals were intraperitoneally injected with N-methyl-N-nitrosourea to induce photoreceptor degeneration. Then optomotor, electroretinogram, and immunostaining were used to test the visual behavior, retinal light responses, and structure. The final ZDE product contained ~30mg/g ZD, which was over 9 times higher than that from the dry fruit of wolfberry. Feeding degenerated mice with ZDE significantly improved the survival of photoreceptors, enhanced the retinal light responses and the visual acuity. Therefore, our ZDE product successfully alleviated retinal morphological and functional degeneration in mouse retina, which may provide a basis for further animal studies for possible applying ZDE as a supplement to treat degenerated photoreceptor in the clinic.
Topics: Animals; Lycium; Retinal Degeneration; Mice; Plant Extracts; Disease Models, Animal; Zeaxanthins; Mice, Inbred C57BL; Photoreceptor Cells, Vertebrate; Electroretinography; Retina; Vision, Ocular; Male; Xanthophylls
PubMed: 38768144
DOI: 10.1371/journal.pone.0302742 -
Experimental Eye Research Jul 2024Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their...
Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2 mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.
Topics: Animals; Retinal Degeneration; Microglia; Mice; Ependymoglial Cells; Electroretinography; Repressor Proteins; Mice, Knockout; Disease Models, Animal; Tomography, Optical Coherence; Prohibitins; Photoreceptor Cells, Vertebrate; Mice, Inbred C57BL; Phagocytosis
PubMed: 38763352
DOI: 10.1016/j.exer.2024.109935 -
Genome Biology May 2024Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited... (Comparative Study)
Comparative Study
BACKGROUND
Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space.
RESULTS
To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4.
CONCLUSIONS
Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.
Topics: Humans; Retinal Pigment Epithelium; Chromatin; Retinal Diseases; Retina; ATP-Binding Cassette Transporters; Animals; Promoter Regions, Genetic; Genetic Loci; Zebrafish; Regulatory Sequences, Nucleic Acid; Genome, Human
PubMed: 38760655
DOI: 10.1186/s13059-024-03250-6 -
Acta Neuropathologica Communications May 2024Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of...
Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of microglia holds promise for mitigating the progression of photoreceptor degeneration. Our previous study has demonstrated that pre-light damage treatment of hyperoside, a naturally occurring flavonol glycoside with antioxidant and anti-inflammatory activities, prevents photooxidative stress-induced photoreceptor degeneration and neuroinflammatory responses in the retina. However, the direct impact of hyperoside on microglia-mediated neuroinflammation during photoreceptor degeneration remains unknown. Upon verifying the anti-inflammatory effects of hyperoside in LPS-stimulated BV-2 cells, our results here further demonstrated that post-light damage hyperoside treatment mitigated the loss of photoreceptors and attenuated the functional decline of the retina. Meanwhile, post-light damage hyperoside treatment lowered neuroinflammatory responses and dampened microglial activation in the illuminated retinas. With respect to microglial activation, hyperoside mitigated the pro-inflammatory responses in DNA-stimulated BV-2 cells and lowered DNA-stimulated production of 2'3'-cGAMP in BV-2 cells. Moreover, hyperoside was shown to directly interact with cGAS and suppress the enzymatic activity of cGAS in a cell-free system. In conclusion, the current study suggests for the first time that the DNA sensor cGAS is a direct target of hyperoside. Hyperoside is effective at mitigating DNA-stimulated cGAS-mediated pro-inflammatory activation of microglia, which likely contributes to the therapeutic effects of hyperoside at curtailing neuroinflammation and alleviating neuroinflammation-instigated photoreceptor degeneration.
Topics: Animals; Microglia; Quercetin; Retinal Degeneration; Mice; Nucleotidyltransferases; Mice, Inbred C57BL; DNA; Cell Line; Photoreceptor Cells, Vertebrate; Male
PubMed: 38755736
DOI: 10.1186/s40478-024-01793-0 -
Cerebral Cortex (New York, N.Y. : 1991) May 2024The effects of hypoxia on brain function remain largely unknown. This study aimed to clarify this issue by visual-stimulated functional magnetic resonance imaging...
The effects of hypoxia on brain function remain largely unknown. This study aimed to clarify this issue by visual-stimulated functional magnetic resonance imaging design. Twenty-three college students with a 30-d high-altitude exposure were tested before, 1 week and 3 months after returning to sea level. Brain functional magnetic resonance imaging and retinal electroretinogram were acquired. One week after returning to sea level, decreased blood oxygenation level dependent in the right lingual gyrus accompanied with increased blood oxygenation level dependent in the frontal cortex and insular cortex, and decreased amplitude of electroretinogram a-wave in right eye; moreover, the bilateral lingual gyri showed increased functional connectivity within the dorsal visual stream pathway, and the blood oxygenation level dependent signals in the right lingual gyrus showed positive correlation with right retinal electroretinogram a-wave. Three months after returning to sea level, the blood oxygenation level dependent signals recovered to normal level, while intensively increased blood oxygenation level dependent signals in a broad of brain regions and decreased retinal electroretinogram were also existed. In conclusion, hypoxic exposure has long-term effects on visual cortex, and the impaired retinal electroretinogram may contribute to it. The increased functional connectivity of dorsal stream may compensate for the decreased function of retinal photoreceptor cells to maintain normal visual function.
Topics: Humans; Male; Young Adult; Magnetic Resonance Imaging; Electroretinography; Female; Neuronal Plasticity; Visual Pathways; Hypoxia; Adult; Oxygen; Visual Cortex; Brain; Photic Stimulation; Retina; Brain Mapping
PubMed: 38752980
DOI: 10.1093/cercor/bhae198 -
FASEB Journal : Official Publication of... May 2024NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective...
NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.
Topics: Animals; Autophagy; NLR Family, Pyrin Domain-Containing 3 Protein; Mice; Apoptosis; Sulfonamides; Inflammasomes; Furans; Ependymoglial Cells; Indenes; Mice, Inbred C57BL; Hypoxia; Cyclic S-Oxides; Sulfones; Photoreceptor Cells, Vertebrate; Heterocyclic Compounds, 4 or More Rings; Photoreceptor Cells; Signal Transduction
PubMed: 38752538
DOI: 10.1096/fj.202301922RR -
BioRxiv : the Preprint Server For... Apr 2024Many inherited retinal diseases target photoreceptors, which transduce light into a neural signal that is processed by the downstream visual system. As photoreceptors...
Many inherited retinal diseases target photoreceptors, which transduce light into a neural signal that is processed by the downstream visual system. As photoreceptors degenerate, physiological and morphological changes to retinal synapses and circuitry reduce sensitivity and increase noise, degrading visual signal fidelity. Here, we pharmacologically targeted the first synapse in the retina in an effort to reduce circuit noise without sacrificing visual sensitivity. We tested a strategy to partially replace the neurotransmitter lost when photoreceptors die with an agonist of receptors that ON bipolars cells use to detect glutamate released from photoreceptors. In mice, which express a photoreceptor mutation that causes retinitis pigmentosa (RP), we found that a low dose of the mGluR6 agonist l-2-amino-4-phosphonobutyric acid (L-AP4) reduced pathological noise induced by photoreceptor degeneration. After making electroretinogram recordings in mice to characterize the developmental time course of visual signal degeneration, we examined effects of L-AP4 on sensitivity and circuit noise by recording light-evoked responses from individual retinal ganglion cells (RGCs). L-AP4 decreased circuit noise evident in RGC recordings without significantly reducing response amplitudes, an effect that persisted over the entire time course of rod photoreceptor degeneration. Subsequent recordings from rod bipolar cells (RBCs) showed that RBCs are more depolarized in retinas, likely contributing to downstream circuit noise and reduced synaptic gain, both of which appear to be ameliorated by hyperpolarizing RBCs with L-AP4. These beneficial effects may reduce pathological circuit remodeling and preserve the efficacy of therapies designed to restore vision.
PubMed: 38746092
DOI: 10.1101/2024.04.30.591881 -
Proceedings of the National Academy of... May 2024Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One...
Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11--retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with and genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.
Topics: Animals; Night Blindness; Eye Diseases, Hereditary; Mice; Rhodopsin; Genetic Diseases, X-Linked; Myopia; Retinal Rod Photoreceptor Cells; Darkness; Transducin; Gene Knock-In Techniques; Disease Models, Animal
PubMed: 38743626
DOI: 10.1073/pnas.2404763121