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1.
J Neuroinflammation ; 21(1): 42, 2024 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-38311721

RESUMO

Diabetic retinopathy (DR) affects about 200 million people worldwide, causing leakage of blood components into retinal tissues, leading to activation of microglia, the resident phagocytes of the retina, promoting neuronal and vascular damage. The microglial receptor, CX3CR1, binds to fractalkine (FKN), an anti-inflammatory chemokine that is expressed on neuronal membranes (mFKN), and undergoes constitutive cleavage to release a soluble domain (sFKN). Deficiencies in CX3CR1 or FKN showed increased microglial activation, inflammation, vascular damage, and neuronal loss in experimental mouse models. To understand the mechanism that regulates microglia function, recombinant adeno-associated viral vectors (rAAV) expressing mFKN or sFKN were delivered to intact retinas prior to diabetes. High-resolution confocal imaging and mRNA-seq were used to analyze microglia morphology and markers of expression, neuronal and vascular health, and inflammatory mediators. We confirmed that prophylactic intra-vitreal administration of rAAV expressing sFKN (rAAV-sFKN), but not mFKN (rAAV-mFKN), in FKNKO retinas provided vasculo- and neuro-protection, reduced microgliosis, mitigated inflammation, improved overall optic nerve health by regulating microglia-mediated inflammation, and prevented fibrin(ogen) leakage at 4 weeks and 10 weeks of diabetes induction. Moreover, administration of sFKN improved visual acuity. Our results elucidated a novel intervention via sFKN gene therapy that provides an alternative pathway to implement translational and therapeutic approaches, preventing diabetes-associated blindness.


Assuntos
Quimiocina CX3CL1 , Diabetes Mellitus , Humanos , Camundongos , Animais , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Microglia/metabolismo , Retina/metabolismo , Diabetes Mellitus/metabolismo , Fatores Imunológicos , Inflamação/metabolismo , Isoformas de Proteínas , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo
2.
Nat Commun ; 15(1): 1124, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38321058

RESUMO

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.


Assuntos
Adenosina , Diabetes Mellitus Experimental , Cinetina , Dinâmica Mitocondrial , Masculino , Camundongos , Humanos , Animais , Neuroproteção , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Mitocôndrias/metabolismo
3.
Int J Mol Sci ; 25(3)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38338800

RESUMO

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.


Assuntos
Degeneração Retiniana , Humanos , Camundongos , Animais , Degeneração Retiniana/metabolismo , Metilnitrosoureia , Retina/metabolismo , Células Fotorreceptoras , Regeneração , Modelos Animais de Doenças , Mamíferos
4.
Int J Mol Sci ; 25(3)2024 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-38338894

RESUMO

This review focuses on the role of small extracellular vesicles in the pathophysiological mechanisms of retinal degenerative diseases. Many of these mechanisms are related to or modulated by the oxidative burden of retinal cells. It has been recently demonstrated that cellular communication in the retina involves extracellular vesicles and that their rate of release and cargo features might be affected by the cellular environment, and in some instances, they might also be mediated by autophagy. The fate of these vesicles is diverse: they could end up in circulation being used as markers, or target neighbor cells modulating gene and protein expression, or eventually, in angiogenesis. Neovascularization in the retina promotes vision loss in diseases such as diabetic retinopathy and age-related macular degeneration. The importance of micro RNAs, either as small extracellular vesicles' cargo or free circulating, in the regulation of retinal angiogenesis is also discussed.


Assuntos
Vesículas Extracelulares , MicroRNAs , Degeneração Retiniana , Humanos , Retina/metabolismo , Degeneração Retiniana/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Oxidativo
5.
Int J Mol Sci ; 25(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38339053

RESUMO

The blood-retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Células-Tronco Mesenquimais , Humanos , Retinopatia Diabética/terapia , Retinopatia Diabética/metabolismo , Pericitos/metabolismo , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Retina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Glucose/metabolismo , Células Cultivadas , Diabetes Mellitus/metabolismo
6.
J Vis Exp ; (203)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38345250

RESUMO

Transplantation of photoreceptor cells and retinal pigment epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into 'adult' rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.


Assuntos
Degeneração Retiniana , Camundongos , Animais , Degeneração Retiniana/cirurgia , Degeneração Retiniana/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transplante de Células/métodos , Visão Ocular
7.
Sci Rep ; 14(1): 3683, 2024 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-38355836

RESUMO

To investigate the association between lactate metabolism and glaucoma, we conducted a multi-institutional cross-sectional clinical study and a retinal metabolomic analysis of mice with elevated intraocular pressure (IOP) induced by intracameral microbead injection. We compared lactate concentrations in serum and aqueous humor in age-matched 64 patients each with primary open-angle glaucoma (POAG) and cataract. Neither serum nor aqueous humor lactate concentrations differed between the two groups. Multiple regression analysis revealed that only body mass index showed a significant positive correlation with serum and aqueous humor lactate concentration in POAG patients (rs = 0.376, P = 0.002, and rs = 0.333, P = 0.007, respectively), but not in cataract patients. L-Lactic acid was one of the most abundantly detected metabolites in mouse retinas with gas chromatography and mass spectrometry, but there were no significant differences among control, 2-week, and 4-week IOP elevation groups. After 4 weeks of elevated IOP, D-glucose and L-glutamic acid ranked as the top two for a change in raised concentration, roughly sevenfold and threefold, respectively (ANOVA, P = 0.004; Tukey-Kramer, P < 0.05). Glaucoma may disrupt the systemic and intraocular lactate metabolic homeostasis, with a compensatory rise in glucose and glutamate in the retina.


Assuntos
Catarata , Glaucoma de Ângulo Aberto , Humanos , Camundongos , Animais , Glaucoma de Ângulo Aberto/metabolismo , Ácido Glutâmico/metabolismo , Ácido Láctico/metabolismo , Estudos Transversais , Cromatografia Gasosa-Espectrometria de Massas , Catarata/metabolismo , Humor Aquoso/metabolismo , Retina/metabolismo , Homeostase , Pressão Intraocular
8.
Nat Commun ; 15(1): 1372, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38355716

RESUMO

Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Animais , Humanos , Barreira Hematorretiniana/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Fenótipo , Dispositivos Lab-On-A-Chip , Vasos Retinianos/metabolismo , Retina/metabolismo , Diabetes Mellitus/metabolismo
9.
Eur J Pharm Biopharm ; 196: 114204, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38302048

RESUMO

In the last few years, mRNA therapeutics experienced a new wave of interest as therapy for retinal diseases. Nevertheless, despite the widespread use of mRNA vaccines in the COVID-19 pandemic, mRNA delivery to the eye is still in its infancy. Recently, our research group has demonstrated that after subretinal and intravitreal delivery of modified mRNA, the number of transfected retinal cells and protein expression per cell remains limited. In this study, we aimed to tackle this limitation by using self-amplifying mRNA (saRNA), which in theory will increase the duration and level of protein expression when only a few mRNA molecules reach their target cells. A one-on-one comparison between modified mRNA and saRNA in two immune-competent human retinal cell types, including Müller cells and retinal pigment epithelial cells, and in immune-deficient BHK-21 cells revealed that saRNA delivery induced an innate immune response blocking its own translation above a certain dose threshold. Removal of double-stranded (ds)RNA byproducts by cellulose-based purification and addition of the innate immune inhibitor B18R remarkably improved translation from saRNA through a reduction in innate immune response. Taken together, when saRNA is applied for retinal disease, the dose should be controlled and measures should be taken to limit immunogenicity.


Assuntos
Pandemias , Retina , Humanos , RNA Mensageiro , Retina/metabolismo , Neurônios/metabolismo
10.
Int J Mol Sci ; 25(4)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38396799

RESUMO

The human retina is a complex anatomical structure that has no regenerative capacity. The pathogenesis of most retinopathies can be attributed to inflammation, with the activation of the inflammasome protein platform, and to the impact of oxidative stress on the regulation of apoptosis and autophagy/mitophagy in retinal cells. In recent years, new therapeutic approaches to treat retinopathies have been investigated. Experimental data suggest that the secretome of mesenchymal cells could reduce oxidative stress, autophagy, and the apoptosis of retinal cells, and in turn, the secretome of the latter could induce changes in mesenchymal cells. Other studies have evidenced that noncoding (nc)RNAs might be new targets for retinopathy treatment and novel disease biomarkers since a correlation has been found between ncRNA levels and retinopathies. A new field to explore is the interaction observed between the ocular and intestinal microbiota; indeed, recent findings have shown that the alteration of gut microbiota seems to be linked to ocular diseases, suggesting a gut-eye axis. To explore new therapeutical strategies for retinopathies, it is important to use proper models that can mimic the complexity of the retina. In this context, retinal organoids represent a good model for the study of the pathophysiology of the retina.


Assuntos
Microbioma Gastrointestinal , Doenças Retinianas , Humanos , Retina/metabolismo , Doenças Retinianas/metabolismo , Inflamação/metabolismo
11.
Int J Mol Sci ; 25(4)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38396985

RESUMO

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Cirurgiões , Humanos , Ratos , Animais , Degeneração Retiniana/patologia , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Retina/metabolismo , Retinite Pigmentosa/metabolismo , Progressão da Doença , Modelos Animais de Doenças
12.
Stem Cell Res Ther ; 15(1): 38, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38336763

RESUMO

BACKGROUND: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally, the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells, a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction. METHODS: We generated NOD1-/--Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow-retina axis. RESULTS: Elevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1-/--Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration, as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations, including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation, and the results further indicated that NOD1 had a protective effect against DR. Mechanistically, loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina, subsequently leading to diminished neutrophil chemoattraction and NETosis. CONCLUSIONS: The results of our study unveil, for the first time, the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation, culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Proteína Adaptadora de Sinalização NOD1 , Degeneração Retiniana , Animais , Camundongos , Medula Óssea/metabolismo , Diabetes Mellitus/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/terapia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Retina/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo
13.
J Neuroinflammation ; 21(1): 56, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38388518

RESUMO

Inherited, age-related, and acute retinal diseases are often exacerbated by an aberrant or excessive activity of the complement system. Consequently, cells not directly affected by an acute event or genetic variants may degenerate, resulting in enhanced visual impairment. The therapeutic potential of supplementation of complement factor H (FH), a key regulator of the complement cascade, is therefore particularly promising in the context of retinal diseases caused by complement activation. In this study, we engineered adeno-associated viruses (AAVs) containing sequences of two truncated human FH variants. The expression of these variants was regulated by the glial fibrillary acidic protein (GFAP) promoter, which is selectively active in gliotic Müller cells. Both FH variants consisted of FH domains 19-20, which were connected to domains 1-4 and 1-7, respectively, by a polyglycine linker. These AAVs were intravitreally injected following ischemic injury of C57BL/6J mouse retinas. We observed transgene expression in gliotic Müller cells and to some extent in astrocytes. The expression correlated directly with damage severity. Interventions resulted in decreased complement activation, accelerated normalization of microglia activity and morphological improvements. Reduced levels of C3 transcripts and C3d protein in conjunction with higher transcript levels of inhibitory regulators like Cfi and Cfh, hinted at attenuated complement activity. This study demonstrates the great potential of complement regulatory gene addition therapy. With further in vivo testing it could be applied to treat a wide range of retinal diseases where no causative therapies are available.


Assuntos
Gliose , Doenças Retinianas , Camundongos , Animais , Humanos , Gliose/metabolismo , Fator H do Complemento/genética , Camundongos Endogâmicos C57BL , Retina/metabolismo
14.
Sci Rep ; 14(1): 4414, 2024 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-38388657

RESUMO

Previous studies have reported increased retinal venous oxygen saturation and decreased retinal blood flow and oxygen metabolism in non-proliferative diabetic retinopathy (NPDR). The current study aimed to determine alterations in both inner retinal oxygen delivery (DO2) and metabolism (MO2) in proliferative DR (PDR) as well as at stages of NPDR. A total of 123 subjects participated in the study and were categorized into five groups: non-diabetic control (N = 32), diabetic with no diabetic retinopathy (NDR, N = 34), mild NPDR (N = 31), moderate to severe NPDR (N = 17), or PDR (N = 9). Multi-modal imaging was performed to measure oxygen saturation and blood flow, which were used for derivation of DO2 and MO2. There were significant associations of groups with DO2 and MO2. DO2 was lower in PDR and not significantly different in NDR and NPDR stages as compared to the non-diabetic control group. MO2 was decreased in PDR and moderate to severe NPDR as compared to the control group, and not significantly reduced in NDR and mild NPDR. The findings demonstrate reductions in both DO2 and MO2 in PDR and MO2 in moderate to severe NPDR, suggesting their potential as biomarkers for monitoring progression and treatment of DR.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Humanos , Retinopatia Diabética/metabolismo , Retina/metabolismo , Oximetria , Biomarcadores , Oxigênio/metabolismo
15.
ACS Chem Neurosci ; 15(4): 735-744, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38324770

RESUMO

Age-related macular degeneration (AMD) is a complex disease in which inflammation is implicated as a key factor but the precise molecular mechanisms are poorly understood. AMD lesions contain an excess of the pro-inflammatory S100A9 protein, but its retinal significance was yet unexplored. S100A9 was shown to be intrinsically amyloidogenic in vitro and in vivo. Here, we hypothesized that the retinal effects of S100A9 are related to its supramolecular conformation. ARPE-19 cultures were treated with native dimeric and fibrillar S100A9 preparations, and cell viability was determined. Wild-type rats were treated intravitreally with the S100A9 solutions in the right eye and with the vehicle in the left. Retinal function was assessed longitudinally by electroretinography (ERG), comparing the amplitudes and configurations for each intervention. Native S100A9 had no impact on cellular viability in vitro or on the retinal function in vivo. Despite dispersed intracellular uptake, fibrillar S100A9 did not decrease ARPE-19 cell viability. In contrast, S100A9 fibrils impaired retinal function in vivo following intravitreal injection in rats. Intriguingly, low-dose fibrillar S100A9 induced contrasting in vivo effects, significantly increasing the ERG responses, particularly over 14 days postinjection. The retinal effects of S100A9 were further characterized by glial and microglial cell activation. We provide the first indication for the retinal effects of S100A9, showing that its fibrils inflicted retinal dysfunction and glial activation in vivo, while low dose of the same assemblies resulted in an unpredicted enhancement of the ERG amplitudes. These nonlinear responses highlight the consequences of self-assembly of S100A9 and provide insight into its pathophysiological and possibly physiological roles in the retina.


Assuntos
Calgranulina B , Degeneração Macular , Ratos , Animais , Calgranulina B/metabolismo , Retina/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Eletrorretinografia , Inflamação/metabolismo , Modelos Animais de Doenças
16.
Cell Mol Neurobiol ; 44(1): 19, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38315298

RESUMO

Retinal vasoactive intestinal peptide amacrine cells (VIP-ACs) play an important role in various retinal light-mediated pathological processes related to different developmental ocular diseases and even mental disorders. It is important to characterize the developmental changes in VIP-ACs to further elucidate their mechanisms of circuit function. We bred VIP-Cre mice with Ai14 and Ai32 to specifically label retinal VIP-ACs. The VIP-AC soma and spine density generally increased, from postnatal day (P)0 to P35, reaching adult levels at P14 and P28, respectively. The VIP-AC soma density curve was different with the VIP-AC spine density curve. The total retinal VIP content reached a high level plateau at P14 but was decreased in adults. From P14 to P16, the resting membrane potential (RMP) became more negative, and the input resistance decreased. Cell membrane capacitance (MC) showed three peaks at P7, P12 and P16. The RMP and MC reached a stable level similar to the adult level at P18, whereas input resistance reached a stable level at P21. The percentage of sustained voltage-dependent potassium currents peaked at P16 and remained stable thereafter. The spontaneous excitatory postsynaptic current and spontaneous inhibitory postsynaptic current frequencies and amplitudes, as well as charge transfer, peaked at P12 to P16; however, there were also secondary peaks at different time points. In conclusion, we found that the second, third and fourth weeks after birth were important periods of VIP-AC development. Many developmental changes occurred around eye opening. The development of soma, dendrite and electrophysiological properties showed uneven dynamics of progression. Cell differentiation may contribute to soma development whereas the changes of different ion channels may play important role for spine development.


Assuntos
Células Amácrinas , Peptídeo Intestinal Vasoativo , Humanos , Camundongos , Animais , Peptídeo Intestinal Vasoativo/metabolismo , Retina/metabolismo , Potenciais da Membrana/fisiologia , Diferenciação Celular
17.
Proc Natl Acad Sci U S A ; 121(7): e2313818121, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38324569

RESUMO

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR's G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the "open" portion of bR's photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR's open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.


Assuntos
Bacteriorodopsinas , Bacteriorodopsinas/metabolismo , Ligantes , Análise Espectral , Retina/metabolismo , Conformação Molecular , Conformação Proteica
18.
Int J Mol Sci ; 25(3)2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38338722

RESUMO

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro.


Assuntos
Corpos Embrioides , Retina , Reprodutibilidade dos Testes , Retina/metabolismo , Organoides , Diferenciação Celular
19.
Nat Commun ; 15(1): 1600, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383453

RESUMO

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.


Assuntos
Oftalmopatias , Multiômica , Humanos , Retina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Genoma , Oftalmopatias/genética , Oftalmopatias/metabolismo
20.
Int J Mol Sci ; 25(3)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38339005

RESUMO

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia-vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia-vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia-neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction.


Assuntos
Quimiocina CX3CL1 , Retinopatia Diabética , Quimiocina CX3CL1/farmacologia , Quimiocina CX3CL1/uso terapêutico , Diabetes Mellitus/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Microglia/metabolismo , Retina/metabolismo , Transdução de Sinais , Complicações do Diabetes/tratamento farmacológico , Animais , Camundongos
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