-
Communications Biology Jun 2021Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and...
Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.
Topics: Ion Transport; Light; Phylogeny; Protein Domains; Proton Pumps; Rhodopsin
PubMed: 34193947
DOI: 10.1038/s42003-021-02326-4 -
Investigative Ophthalmology & Visual... Sep 2023Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of...
Kallistatin Deficiency Induces the Oxidative Stress-Related Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells: A Novel Protagonist in Age-Related Macular Degeneration.
PURPOSE
Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of age-related macular degeneration (AMD). Kallistatin (KAL) is a secreted protein with an antioxidative stress effect. However, the relationship between KAL and EMT in RPE has not been determined. Therefore we aimed to explore the impact and mechanism of KAL in oxidative stress-induced EMT of RPE.
METHODS
Sodium iodate (SI) was injected intraperitoneally to construct the AMD rat model and investigate the changes in RPE morphology and KAL expression. KAL knockout rats and KAL transgenic mice were used to explain the effects of KAL on EMT and oxidative stress. In addition, Snail overexpressed adenovirus and si-RNA transfected ARPE19 cells to verify the involvement of Snail in mediating KAL-suppressed EMT of RPE.
RESULTS
AMD rats induced by SI expressed less KAL in the retina, and KAL knockout rats showed RPE dysfunction spontaneously where EMT and reactive oxygen species (ROS) production increased in RPE. In contrast, KAL overexpression attenuated EMT and ROS levels in RPE, even in TGF-β treatment. Mechanistically, Snail reversed the beneficial effect of KAL on EMT and ROS reduction. Moreover, KAL ameliorated SI-induced AMD-like pathological changes.
CONCLUSIONS
Our findings demonstrated that KAL inhibits oxidative stress-induced EMT by downregulating the transcription factor Snail. Herein, KAL knockout rats may be an appropriate animal model for observing spontaneous RPE dysfunction for AMD-like retinopathy, and KAL may represent a novel therapeutic target for treating dry AMD.
Topics: Animals; Mice; Rats; Epithelial Cells; Epithelial-Mesenchymal Transition; Geographic Atrophy; Macular Degeneration; Mice, Transgenic; Oxidative Stress; Reactive Oxygen Species; Retinal Pigments; Serpins
PubMed: 37682567
DOI: 10.1167/iovs.64.12.15 -
Stem Cell Research & Therapy Jun 2022Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular...
BACKGROUND
Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics.
METHODS
We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins.
RESULTS
We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p and MELAS iPSC-derived RPE cells.
CONCLUSIONS
Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.
Topics: Autophagy; DNA, Mitochondrial; Energy Metabolism; Epithelial Cells; Humans; Induced Pluripotent Stem Cells; MELAS Syndrome; Macular Degeneration; Mitochondria; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 35715869
DOI: 10.1186/s13287-022-02937-6 -
Philosophical Transactions of the Royal... Jan 2016Vision allows animals to detect spatial differences in environmental light levels. High-resolution image-forming eyes evolved from low-resolution eyes via increases in... (Review)
Review
Vision allows animals to detect spatial differences in environmental light levels. High-resolution image-forming eyes evolved from low-resolution eyes via increases in photoreceptor cell number, improvements in optics and changes in the neural circuits that process spatially resolved photoreceptor input. However, the evolutionary origins of the first low-resolution visual systems have been unclear. We propose that the lowest resolving (two-pixel) visual systems could initially have functioned in visual phototaxis. During visual phototaxis, such elementary visual systems compare light on either side of the body to regulate phototactic turns. Another, even simpler and non-visual strategy is characteristic of helical phototaxis, mediated by sensory-motor eyespots. The recent mapping of the complete neural circuitry (connectome) of an elementary visual system in the larva of the annelid Platynereis dumerilii sheds new light on the possible paths from non-visual to visual phototaxis and to image-forming vision. We outline an evolutionary scenario focusing on the neuronal circuitry to account for these transitions. We also present a comprehensive review of the structure of phototactic eyes in invertebrate larvae and assign them to the non-visual and visual categories. We propose that non-visual systems may have preceded visual phototactic systems in evolution that in turn may have repeatedly served as intermediates during the evolution of image-forming eyes.
Topics: Animals; Biological Evolution; Eye; Gene Expression Regulation; Opsins; Photoreceptor Cells
PubMed: 26598725
DOI: 10.1098/rstb.2015.0042 -
Cell Death & Disease Sep 2022Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their...
Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their limited differentiation capacity in vivo attenuates their therapeutic function. Therefore, the differentiation of MSCs into retinal pigment epithelial (RPE) cells in vitro and their subsequent transplantation into the subretinal space is expected to improve the outcome of cell therapy. Here, we transdifferentiated human umbilical cord MSCs (hUCMSCs) into induced RPE (iRPE) cells using a cocktail of five transcription factors (TFs): CRX, NR2E1, C-MYC, LHX2, and SIX6. iRPE cells exhibited RPE specific properties, including phagocytic ability, epithelial polarity, and gene expression profile. In addition, high expression of PTPN13 in iRPE cells endows them with an epithelial-to-mesenchymal transition (EMT)-resistant capacity through dephosphorylating syntenin1, and subsequently promoting the internalization and degradation of transforming growth factor-β receptors. After grafting into the subretinal space of the sodium iodate-induced rat AMD model, iRPE cells demonstrated a better therapeutic function than hUCMSCs. These results suggest that hUCMSC-derived iRPE cells may be promising candidates to reverse AMD pathophysiology.
Topics: Animals; Epithelial Cells; Humans; LIM-Homeodomain Proteins; Macular Degeneration; Mesenchymal Stem Cells; Rats; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Pigments; Transcription Factors; Umbilical Cord
PubMed: 36096985
DOI: 10.1038/s41419-022-05199-5 -
Journal of Anatomy Aug 2023Retinal pigment epithelium (RPE) is a specialized pigmented monolayer dedicated to retinal support and protection. Given the fact that photoreceptor outer segments are...
Retinal pigment epithelium (RPE) is a specialized pigmented monolayer dedicated to retinal support and protection. Given the fact that photoreceptor outer segments are the primary energy resource of RPE metabolism, it follows that, when photoreceptor function is compromised, RPE cells are impaired and vice versa. In retinitis pigmentosa (RP), genetic mutations lead to a massive degeneration of photoreceptors but only few studies have addressed systematically the consequences of rod and cone death on RPE cells, which, among others, undergo an abnormal organization of tight junctions (TJs) and a compromised barrier function. The biological mechanisms driving these barrier reorganizations are largely unknown. Studies aimed at addressing general and mutation-independent changes of the RPE in RP are relevant to reveal new pathogenic mechanisms of this heterogeneous family of diseases and prospectively develop effective therapeutic strategies. Here, we take advantage of a mouse model of RP in which retinal degeneration is spatially restricted to investigate a possible involvement of inflammatory responses in RPE remodeling. By immunostaining for Zona Occludens-1 (ZO-1), a structural and functional marker of TJs with pleiotropic functions, we found a partial rescue of TJs organization following local restoration of retinal organization, revealing that TJs structure can recover. Since lack of ZO-1 from TJs can alter cell density, we counted RPE cells without finding any differences between degenerated and controls animals, indicating preservation of RPE cells. However, we found an increased number of immune cells adhering to the RPE apical surface and a spatial correlation with areas of abnormal ZO-1 distribution. This suggests that inflammatory processes following photoreceptor degeneration can be responsible for TJs alterations during RP progression and deserve further investigation.
Topics: Mice; Animals; Rhodopsin; Retina; Retinal Degeneration; Retinitis Pigmentosa; Retinal Pigment Epithelium; Epithelial Cells
PubMed: 35428980
DOI: 10.1111/joa.13667 -
Molecules and Cells Jul 2023Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of...
Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of anti-vascular endothelial growth factor are invasive, and repetitive injections are also accompanied by a risk of intraocular infection. The pathogenic mechanism of AMD is still not completely understood, but a multifactorial mechanism that combines genetic predisposition and environmental factors, including cellular senescence, has been suggested. Cellular senescence refers to the accumulation of cells that stop dividing due to the presence of free radicals and DNA damage. Characteristics of senescent cells include nuclear hypertrophy, increased levels of cell cycle inhibitors such as p16 and p21, and resistance to apoptosis. Senolytic drugs remove senescent cells by targeting the main characteristics of these cells. One of the senolytic drugs, ABT-263, which inhibits the antiapoptotic functions of Bcl-2 and Bcl-xL, may be a new treatment for AMD patients because it targets senescent retinal pigment epithelium (RPE) cells. We proved that it selectively kills doxorubicin (Dox)-induced senescent ARPE-19 cells by activating apoptosis. By removing senescent cells, the expression of inflammatory cytokines was reduced, and the proliferation of the remaining cells was increased. When ABT-263 was orally administered to the mouse model of senescent RPE cells induced by Dox, we confirmed that senescent RPE cells were selectively removed and retinal degeneration was alleviated. Therefore, we suggest that ABT-263, which removes senescent RPE cells through its senolytic effect, has the potential to be the first orally administered senolytic drug for the treatment of AMD.
Topics: Animals; Mice; Retinal Degeneration; Senotherapeutics; Antineoplastic Agents; Macular Degeneration; Apoptosis; Epithelial Cells; Retinal Pigments; Cellular Senescence
PubMed: 37222160
DOI: 10.14348/molcells.2023.2188 -
International Journal of Molecular... Jan 2023Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. With aging and the accumulated effects of...
Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. With aging and the accumulated effects of environmental stress, retinal pigment epithelial (RPE) cells are particularly susceptible to oxidative damage, which can lead to retinal degeneration. However, the underlying molecular mechanisms of how RPE responds and progresses under oxidative damage are still largely unknown. Here, we reveal that exogenous oxidative stress led to ferroptosis characterized by Fe accumulation and lipid peroxidation in RPE cells. Glutathione specific gamma-glutamylcyclotransferase 1 (), as a component of the unfolded protein response (UPR) pathway, plays a pivotal role in oxidative-stress-induced cell ferroptosis via the regulation of glutathione depletion. These results indicate the biological significance of as a novel contributor of oxidative-stress-induced ferroptosis in RPE, suggesting its potential role in AMD.
Topics: Aged; Humans; Epithelial Cells; Ferroptosis; Glutathione; Macular Degeneration; Oxidative Stress; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 36675091
DOI: 10.3390/ijms24021582 -
Biochemical and Biophysical Research... Aug 2022Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and...
Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and in retinal pigment epithelial (ARPE-19) cells. We hypothesized that these redoxins regulate cellular functions and signaling circuits such as cell proliferation, Wnt signaling and VEGF release that have been correlated to the pathophysiology of AMD. ARPE-19 cells were transfected with specific siRNAs to silence the expression of Grx1 and Grx2 and were analyzed for proliferation/viability, migration capacity, β-catenin activation, and VEGF release. An active site-mutated C-X-X-S Grx1 was utilized to trap interacting proteins present in ARPE-19 cell extracts. In both, AMD retinas and in ARPE-19 cells incubated under hypoxia/reoxygenation conditions, Grx1 showed an increased nuclear localization. Grx1-silenced ARPE-19 cells showed a significantly reduced proliferation and migration rate. Our trapping approach showed that Grx1 interacts with β-catenin in a dithiol-disulfide exchange reaction. Knock-down of Grx1 led to a reduction in both total and active β-catenin levels. These findings add redox control to the regulatory mechanisms of β-catenin signaling in the retinal pigment epithelium and open the door to novel therapeutic approaches in AMD that is currently treated with VEGF-inhibitors.
Topics: Cell Proliferation; Epithelial Cells; Glutaredoxins; Humans; Macular Degeneration; Retinal Pigment Epithelium; Retinal Pigments; Signal Transduction; Vascular Endothelial Growth Factor A; beta Catenin
PubMed: 35714567
DOI: 10.1016/j.bbrc.2022.06.030 -
Investigative Ophthalmology & Visual... Oct 2023Proteopathy is believed to contribute to age-related macular degeneration (AMD). Much research indicates that AMD begins in the retinal pigment epithelium (RPE), which...
PURPOSE
Proteopathy is believed to contribute to age-related macular degeneration (AMD). Much research indicates that AMD begins in the retinal pigment epithelium (RPE), which is associated with formation of extracellular drusen, a clinical hallmark of AMD. Human RPE produces a drusen-associated abnormal protein, the exon Ⅵ-skipping splice isoform of retinal G protein-coupled receptor (RGR-d). In this study, we investigate the detrimental effects of RGR-d on cultured cells and mouse retina.
METHODS
ARPE-19 cells were stably infected by lentivirus overexpressing RGR or RGR-d and were treated with MG132, sometimes combined with or without endoplasmic reticulum (ER) stress inducer, tunicamycin. RGR and RGR-d protein expression, degeneration pathway, and potential cytotoxicity were explored. Homozygous RGR-d mice aged 8 or 14 months were fed with a high-fat diet for 3 months and then subjected to ocular examination and histopathology experiments.
RESULTS
We confirm that RGR-d is proteotoxic under various conditions. In ARPE-19 cells, RGR-d is misfolded and almost completely degraded via the ubiquitin-proteasome system. Unlike normal RGR, RGR-d increases ER stress, triggers the unfolded protein response, and exerts potent cytotoxicity. Aged RGR-d mice manifest disrupted RPE cell integrity, apoptotic photoreceptors, choroidal deposition of complement C3, and CD86+CD32+ proinflammatory cell infiltration into retina and RPE-choroid. Furthermore, the AMD-like phenotype of RGR-d mice can be aggravated by a high-fat diet.
CONCLUSIONS
Our study confirmed the pathogenicity of the RGR splice isoform and corroborated a significant role of proteopathy in AMD. These findings may contribute to greater comprehension of the multifactorial causes of AMD.
Topics: Animals; Humans; Mice; Exons; Macular Degeneration; Opsins; Protein Isoforms; Retina; Receptors, G-Protein-Coupled; Eye Proteins
PubMed: 37883094
DOI: 10.1167/iovs.64.13.41