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Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.Molecular Therapy. Nucleic Acids Jun 2024Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy...
Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.
PubMed: 38868364
DOI: 10.1016/j.omtn.2024.102222 -
Cureus May 2024Objective In this study, we aimed to evaluate the choroidal thickness in patients with unilateral strabismic amblyopia by using spectral domain-enhanced depth...
Objective In this study, we aimed to evaluate the choroidal thickness in patients with unilateral strabismic amblyopia by using spectral domain-enhanced depth imaging-optical coherence tomography (SD-EDI-OCT) (Heidelberg Engineering GmbH, Heidelberg, Germany). Methods Twenty-five children with strabismic amblyopia and 20 age- and sex-matched healthy controls were included in this study. Seven sections were obtained, each comprising 25 repetitive images from each section at 200-micron intervals, and measurements were taken from nine different points at vertical and horizontal lines (1 and 3 mm from the subfoveal, superior, inferior, temporal, and nasal regions), centered on the fovea, using SD-EDI-OCT. Choroidal thickness values were obtained by measuring the distance between the basal border of the retinal pigment epithelium and the choroidoscleral border. The Mann-Whitney U test was used to compare choroidal thickness between the amblyopic and the control groups. Results The mean age of children with amblyopia and that of controls were 8.4 ±2.7 and 9.9 ±3.3 years, respectively (p=0.120). The mean subfoveal choroidal thickness was 372.8 ±78.9 μm in amblyopic eyes and 372.4 ±79.3 μm in the fellow eyes, both of which were thicker than the control eyes (310.9 ±76.3 μm; p<0.05 for each). Similarly, the mean values for the choroidal thickness of the amblyopic children at 1 mm nasal (320 ±86 μm), 1 mm superior (363 ±70 μm), and 3 mm superior (336 ±62 μm) were also significantly thicker than those of the corresponding control eyes (p<0.05 for each). There was a negative correlation between the subfoveal choroidal thickness and axial length (r=-0.332, p=0.005). There were no correlations between the choroidal thickness, age, and visual acuity. Conclusions The choroidal thicknesses of strabismic and fellow eyes were similar in patients with strabismic amblyopia. However, the choroidal thickness of both eyes in strabismic children was significantly thicker than those of the healthy controls. Emmetropization may be defective in both eyes of strabismic amblyopic patients.
PubMed: 38868277
DOI: 10.7759/cureus.60219 -
Proceedings of the National Academy of... Jun 2024Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive...
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
Topics: Animals; Mitochondria; Mice; Oxidoreductases; Retinal Pigment Epithelium; Plant Proteins; Mitochondrial Proteins; Ciona intestinalis; Ubiquinone; Retinal Degeneration; Photoreceptor Cells, Vertebrate
PubMed: 38865272
DOI: 10.1073/pnas.2402384121 -
Investigative Ophthalmology & Visual... Jun 2024N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl...
PURPOSE
N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE.
METHODS
Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, β-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography.
RESULTS
IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo.
CONCLUSIONS
Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.
Topics: Retinal Pigment Epithelium; Animals; Mice; PAX6 Transcription Factor; Homeostasis; Mice, Inbred C57BL; Otx Transcription Factors; RNA-Binding Proteins; Phagocytosis; Flow Cytometry; Gene Expression Regulation; Tomography, Optical Coherence; Electroretinography; Cells, Cultured
PubMed: 38861275
DOI: 10.1167/iovs.65.6.17 -
Oxford Medical Case Reports Jun 2024Pigmentary retinal dystrophy (PRD) is a group of inherited disorders involving the progressive degeneration of rod and cone photoreceptors and the retinal pigment...
Pigmentary retinal dystrophy (PRD) is a group of inherited disorders involving the progressive degeneration of rod and cone photoreceptors and the retinal pigment epithelium (RPE), which can progress to pigmentary retinopathy (PR). We present a case of PRD in a female pediatric patient who has pathogenic variants in the PRPH2 and PEX1 genes. The patient has associated macular edema and secondary visual impairment. Treatment has included serial dexamethasone intravitreal implant injections and topical dorzolamide. The PEX1 gene mutation is associated with peroxisome biogenesis disorder-Zellweger syndrome spectrum (PBD-ZSS) and resulting retinal dystrophies. The PRPH2 mutation may play a role in macular edema and PRD, as it is implicated in macular degeneration, choroid defects, and photoreceptor dysfunction. In this case, we review multiple gene mutations playing potential etiologic roles for PRD and discuss care management.
PubMed: 38860019
DOI: 10.1093/omcr/omae067 -
Journal of Physiological Investigation May 2024Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer...
Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.
Topics: Curcumin; Diabetic Retinopathy; Retinal Pigment Epithelium; Humans; Cellular Senescence; Tight Junctions; Animals; Male; Apoptosis; Cell Survival; Blood-Retinal Barrier; Mice, Inbred C57BL; Mice
PubMed: 38857204
DOI: 10.4103/ejpi.EJPI-D-23-00035 -
Biomedical Optics Express May 2024Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the...
Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.
PubMed: 38855698
DOI: 10.1364/BOE.511806 -
Results in Chemistry Dec 2023γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of...
Cysteinyl leukotriene-like metabolites are generated in retinal pigment epithelial cells through glutathionylation/reduction of an oxidatively truncated fragment of arachidonate.
γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C. We now report that exposure of human retinal pigment epithelial cells to CoCl, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD and LTE. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard C N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling.
PubMed: 38855016
DOI: 10.1016/j.rechem.2023.100995 -
BioRxiv : the Preprint Server For... Jun 2024Mutations in progranulin ( ) cause frontotemporal dementia ( -FTD) due to deficiency of the pleiotropic protein progranulin. -FTD exhibits diverse pathologies...
Mutations in progranulin ( ) cause frontotemporal dementia ( -FTD) due to deficiency of the pleiotropic protein progranulin. -FTD exhibits diverse pathologies including lysosome dysfunction, lipofuscinosis, microgliosis, and neuroinflammation. Yet, how progranulin loss causes disease remains unresolved. Here, we report that non-invasive retinal imaging of -FTD patients revealed deficits in photoreceptors and the retinal pigment epithelium (RPE) that correlate with cognitive decline. Likewise, mice exhibit early RPE dysfunction, microglial activation, and subsequent photoreceptor loss. Super-resolution live imaging and transcriptomic analyses identified RPE mitochondria as an early driver of retinal dysfunction. Loss of mitochondrial fission protein 1 (MTFP1) in RPE causes mitochondrial hyperfusion and bioenergetic defects, leading to NF-kB-mediated activation of complement C3a-C3a receptor signaling, which drives further mitochondrial hyperfusion and retinal inflammation. C3aR antagonism restores RPE mitochondrial integrity and limits subretinal microglial activation. Our study identifies a previously unrecognized mechanism by which progranulin modulates mitochondrial integrity and complement-mediated neuroinflammation.
PubMed: 38854134
DOI: 10.1101/2024.05.29.595206 -
BioRxiv : the Preprint Server For... Jun 2024Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in...
Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.
PubMed: 38853929
DOI: 10.1101/2024.05.31.596474