RESUMO
OBJECTIVES: MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. MATERIALS AND METHODS: A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. CONCLUSIONS: Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.
Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Degeneração Retiniana , Animais , Humanos , Degeneração Retiniana/metabolismo , Recoverina/metabolismo , Medula Óssea/metabolismo , Células Fotorreceptoras/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , Proteína Quinase C/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Anti-recoverin antibodies are typically found in cancer-associated retinopathy or autoimmune diseases. We present a case of anti-recoverin positive cerebellar syndrome without any signs of malignancy or retinopathy. The patient was treated with steroids and intravenous immunoglobulins, resulting in improvements in both cognitive and motor symptoms.
Assuntos
Doenças Autoimunes , Doenças Retinianas , Humanos , Recoverina , Retina/patologia , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/etiologiaRESUMO
Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.
Assuntos
Cálcio , Proteínas Sensoras de Cálcio Neuronal , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Cálcio/metabolismo , Retina/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Recoverina/genética , Recoverina/metabolismoRESUMO
To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.
Assuntos
Antineoplásicos , Humanos , Recoverina , Células A549 , Sobrevivência Celular , Tripsina/farmacologia , Antineoplásicos/farmacologia , Receptores Acoplados a Proteínas G/genética , Esferoides CelularesRESUMO
PURPOSE: To evaluate plasma antiretinal autoantibody (ARA) profiling and diagnostic efficacy for autoimmune retinopathy (AIR). DESIGN: A multicenter, diagnostic evaluation study. METHODS: Forty-nine patients with a clinical diagnosis of AIR, disease controls including 20 patients with retinitis pigmentosa (RP), and 30 normal controls were included. Plasma samples from patients were analyzed for the presence of 6 ARAs, including recoverin, α-enolase, carbonic anhydrase II, heat shock protein 60, aldolase C, and cone-rod homeobox/cone-rod retinal dystrophy 2 using western blotting. RESULTS: Autoantibody detection rates against cone-rod homeobox/cone-rod retinal dystrophy 2, heat shock protein 60, and aldolase C in AIR were 67.3%, 40.8%, and 42.9%, respectively, which were higher than those in RP and normal controls (P < .001, P < .001, and P = .007, respectively), but recoverin, α-enolase, and carbonic anhydrase II were not different from other control groups (P = .117, P = .774, and P = .467, respectively). Among ARAs, antirecoverin antibody was the most specific, as it was found in 3 (6.1%) patients with AIR and none of the control groups. As the number of detected ARAs increased, the probability of AIR increased (odds ratio: 1.913; P < .001; 95% confidence interval: 1.456-2.785). The positive number of ARAs was significantly higher when photoreceptor disruption was observed on optical coherence tomography, or severe dysfunction was observed in electroretinography (P = .022 and P = .029, respectively). CONCLUSIONS: The profiles of ARAs in the AIR group were different from those in the RP and normal controls. The higher number of positive ARAs suggests a higher possibility of AIR diagnosis. ARAs should be used as adjunct tools for the clinical diagnosis of AIR.
Assuntos
Doenças Autoimunes , Distrofias de Cones e Bastonetes , Doenças Retinianas , Retinose Pigmentar , Humanos , Doenças Autoimunes/diagnóstico , Autoanticorpos , Doenças Retinianas/diagnóstico , Recoverina , Anidrase Carbônica II , Chaperonina 60 , Frutose-Bifosfato Aldolase , Eletrorretinografia , Retinose Pigmentar/diagnóstico , Fosfopiruvato HidrataseRESUMO
Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.
Assuntos
Cálcio , Caveolina 1 , Recoverina/metabolismo , Cálcio/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Oxirredução , Dissulfetos/metabolismo , Visão Ocular , Compostos de SulfidrilaRESUMO
Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Arrestinas , Biomarcadores Tumorais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/patologia , Receptor Quinase 1 Acoplada a Proteína G , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/patologia , Recoverina , Retina , TransducinaRESUMO
When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Iris , Neurônios Retinianos , Animais , Camundongos , Calbindinas/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Iris/citologia , Camundongos Endogâmicos DBA , Recoverina/metabolismo , Neurônios Retinianos/metabolismoRESUMO
Known as a common malignant tumor among children, retinoblastoma (RB) is highly malignant and has poor prognosis, damages children's vision and degrades quality of life. To identify a potential molecular mechanism of RB, we conducted this study on legumain (LGMN), which is highly expressed in multiple tumors. In this study, we found that LGMN was significantly upregulated in RB cells and was positively expressed in RB tissues. We confirmed that LGMN overexpression (LGMN-OE) can promote RB cell proliferation and inhibit cell apoptosis through CCK8 experiments and flow cytometry. In addition, real-time quantitative polymerase chain reaction (RTâqPCR) and Western blot results showed that LGMN-OE could regulate the expression of epithelial-mesenchymal transformation-related genes and proteins, related to tumor invasion and metastasis. Moreover, after LGMN knock down, the result was the opposite., RNA sequence analysis revealed 1159 differentially expressed genes between LGMN-OE and the negative control (NCOE), of which 564 were upregulated and 595 were downregulated. The first 10 genes were verified by RTâqPCR based on P value and fold change. Interestingly, we found that LGMN could regulate the expression of recoverin (RCVRN)through a gene responsible for cancer-related retinopathy. We also screened and verified that LGMN partially activated the PI3K/AKT pathway in RB. Furthermore, we evaluated the effect of legumain inhibitors (e.g., esomeprazole) on RB, and the results suggest that esomeprazole may provide a reference for the clinical adjuvant treatment of RB. In conclusion, legumain can serve as an attractive target for RB therapy and hopefully provide new insights and ideas for the development of targeted drugs and precise personalized clinical therapy.
Assuntos
MicroRNAs , Neoplasias da Retina , Retinoblastoma , Criança , Humanos , Retinoblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Recoverina/genética , Recoverina/metabolismo , Recoverina/farmacologia , Esomeprazol/farmacologia , Qualidade de Vida , Regulação Neoplásica da Expressão Gênica , Movimento Celular , MicroRNAs/genética , Transdução de Sinais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias da Retina/patologiaRESUMO
Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma.
Assuntos
Cálcio , Proteínas Sensoras de Cálcio Neuronal , Cálcio/metabolismo , Motivos EF Hand , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Ligação Proteica/fisiologia , Recoverina/química , Recoverina/metabolismo , Zinco/metabolismoAssuntos
COVID-19 , Doenças Retinianas , Anticorpos , Humanos , RNA Viral , Recoverina , SARS-CoV-2 , SíndromeRESUMO
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic autosomal recessive disorder caused by mutation in the autoimmune regulator (AIRE) gene. Patients usually are diagnosed at ages between 5 and 15 years when they show 3 or more manifestations, most typically mucocutaneous candidiasis, Addison's disease, and hypoparathyroidism. APECED-associated hepatitis (APAH) develops in only 10% to 40% of patients, with severity varying from subclinical chronic active hepatitis to potentially fatal acute liver failure (ALF). Ocular abnormalities are fairly common, most often keratopathy but sometimes retinopathy. Here we report a 2-year-old Japanese girl with an AIRE gene mutation who developed APAH with ALF, preceded by autoimmune retinopathy associated with anti-recoverin antibody before major symptoms suggested a diagnosis of APECED. Intravenous pulse methylprednisolone therapy followed by a corticosteroid combined with azathioprine treatment resolved ALF and achieved control of APAH. To our knowledge, our patient is the youngest reported to have ALF resulting from an AIRE gene mutation. Pulse methylprednisolone induction therapy followed by treatment with corticosteroid plus azathioprine may well be effective in other children with APAH and AIRE gene mutations.
Assuntos
Corticosteroides/administração & dosagem , Doenças Autoimunes/tratamento farmacológico , Falência Hepática Aguda/tratamento farmacológico , Metilprednisolona/administração & dosagem , Mutação , Poliendocrinopatias Autoimunes/tratamento farmacológico , Doenças Retinianas/tratamento farmacológico , Fatores de Transcrição/genética , Administração Intravenosa , Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Azatioprina/administração & dosagem , Pré-Escolar , Quimioterapia Combinada , Feminino , Predisposição Genética para Doença , Humanos , Imunossupressores/administração & dosagem , Falência Hepática Aguda/diagnóstico , Falência Hepática Aguda/genética , Falência Hepática Aguda/imunologia , Fenótipo , Poliendocrinopatias Autoimunes/diagnóstico , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Pulsoterapia , Recoverina/imunologia , Doenças Retinianas/diagnóstico , Doenças Retinianas/genética , Doenças Retinianas/imunologia , Resultado do Tratamento , Proteína AIRERESUMO
BACKGROUND: Tonsillectomy and steroid pulse therapy (TSP) for immunoglobulin A nephropathy (IgAN) is frequently employed in many Japanese institutions; however, performing this invasive treatment in patients with mild IgAN is controversial. This study aimed to evaluate the appropriate treatment for IgAN patients with mild proteinuria. METHODS: In this retrospective cohort analysis, 122 IgAN patients with mild proteinuria (0.5-1.0 g/day) and estimated glomerular filtration rate of ≥ 60 mL/min/1.73 m2 were classified into three groups as follows: patients treated with TSP (n = 32), oral prednisolone (oPSL, n = 33), and conservative therapy (CONS, n = 47). The clinical and histological backgrounds, 5-year remission rates of urinary findings, and 10-year renal survival rates were analyzed. RESULTS: The backgrounds were similar among the three groups. The remission rates of hematuria, proteinuria, and both were significantly higher for TSP and oPSL than for CONS; however, they were similar for TSP and oPSL. In the multivariate Cox regression analysis, TSP and oPSL were independent factors for the remission of urinary findings compared with CONS; however, the relapse rates of urinary abnormalities were similar among the three groups. No patient progressed to end-stage renal disease (ESRD) within 10 years. Adverse effects of corticosteroid therapy were significantly more frequent in oPSL than in TSP. CONCLUSION: In IgAN patients with mild proteinuria and stable renal function, similar to oPSL, TSP showed higher remission rates of hematuria and/or proteinuria than CONS, and no case progressed to ESRD regardless of the treatment methods. Therefore, appropriate treatments should be carefully considered for each patient.
Assuntos
Anti-Inflamatórios/uso terapêutico , Glomerulonefrite por IGA/fisiopatologia , Glomerulonefrite por IGA/terapia , Prednisolona/uso terapêutico , Tonsilectomia , Adulto , Anti-Inflamatórios/administração & dosagem , Tratamento Conservador , Progressão da Doença , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite por IGA/complicações , Hematúria/etiologia , Humanos , Masculino , Prednisolona/administração & dosagem , Prognóstico , Proteinúria/etiologia , Recoverina , Indução de Remissão , Estudos Retrospectivos , Fatores de Tempo , Adulto JovemRESUMO
Toxoplasma gondii infection can trigger autoreactivity by different mechanisms. In the case of ocular toxoplasmosis, disruption of the blood-retinal barrier may cause exposure of confined retinal antigens such as recoverin. Besides, cross-reactivity can be induced by molecular mimicry of parasite antigens like HSP70, which shares 76% identity with the human ortholog. Autoreactivity can be a determining factor of clinical manifestations in the eye and in the central nervous system. We performed a prospective observational study to determine the presence of autoantibodies against recoverin and HSP70 by indirect ELISA in the serum of 65 patients with ocular, neuro-ophthalmic and congenital cerebral toxoplasmosis. We found systemic autoantibodies against recoverin and HSP70 in 33.8% and 15.6% of individuals, respectively. The presence of autoantibodies in cases of OT may be related to the severity of clinical manifestations, while in cases with CNS involvement they may have a protective role. Unexpectedly, anti-recoverin antibodies were found in patients with cerebral involvement, without ocular toxoplasmosis; therefore, we analyzed and proved cross-reactivity between recoverin and a brain antigen, hippocalcin, so the immunological phenomenon occurring in one immune-privileged organ (e.g. the central nervous system) could affect the environment of another (egg. the eye).
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Interações Hospedeiro-Parasita/imunologia , Toxoplasmose Cerebral/imunologia , Toxoplasmose Congênita/imunologia , Toxoplasmose Ocular/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Feminino , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Hipocalcina/química , Hipocalcina/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Recoverina/química , Recoverina/imunologia , Toxoplasma/imunologia , Toxoplasmose Cerebral/diagnóstico , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Congênita/diagnóstico , Toxoplasmose Congênita/parasitologia , Toxoplasmose Ocular/diagnóstico , Toxoplasmose Ocular/parasitologia , Adulto JovemRESUMO
IMPORTANCE: The clinical utility of most antiretinal antibodies (retina antibodies) currently available for testing remains unclear and unproven. Despite this, the presence of retinal antibodies is included in current diagnostic autoimmune retinopathy criteria. OBJECTIVE: To evaluate the clinical significance of comprehensive retinal antibody evaluations currently offered in North America. DESIGN, SETTING, AND PARTICIPANTS: In this cross-sectional study, 14 patients without autoimmune retinopathy were recruited into the Mayo Clinic Neuroimmunology Biorepository for this study between January 1, 2019, and October 1, 2019. These serum samples without autoimmune retinopathy were sent in masked fashion to a Clinical Laboratory Improvement Amendments-certified laboratory. Using similar methods, the Mayo Clinic Neuroimmunology Research Laboratory independently assessed the same sample to ascertain reproducibility of the findings. MAIN OUTCOMES AND MEASURES: Results of the autoimmune retinopathy and cancer-associated retinopathy panels. RESULTS: Thirteen of 14 (93%; 95% CI, 66%-100%) serum samples tested positive for retinal antibodies, with a median of 5 retinal antibodies (range, 0-8) per patient at the Clinical Laboratory Improvement Amendments-certified laboratory, which provides a specificity of 7% (95% CI, 0%-34%). Confirmatory immunohistochemistry staining in human retina was present in 12 of 14 samples (86%). α-Enolase was found in 9 (64%). The only retinal antibody not present was recoverin. These nonspecific retinal antibody results were replicated at the Mayo Clinic Laboratory on Western blot using pig retina proteins as substrate. CONCLUSIONS AND RELEVANCE: The presence of retinal antibodies in 93% of the patients without autoimmune retinopathy indicates a lack of specificity and that most detectable retinal antibodies have limited clinical relevance in the evaluation of patients for suspected autoimmune retinopathy. Current retinal antibody testing, other than recoverin, should be interpreted with caution, especially for cases of low clinical suspicion. The poor specificity is important to recognize to prevent the potentially unnecessary commencement of systemic immunosuppressants that may result in significant extraocular adverse effects. Identification of biomarkers that have a high predictive value for inflammatory or autoimmune retinal diseases is needed to move the field forward.
Assuntos
Doenças Autoimunes , Doenças Retinianas , Animais , Autoanticorpos , Autoantígenos , Doenças Autoimunes/diagnóstico , Estudos Transversais , Humanos , Recoverina , Reprodutibilidade dos Testes , Retina , Doenças Retinianas/diagnóstico , SuínosRESUMO
Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.
Assuntos
Fenômenos Eletrofisiológicos , Células-Tronco Pluripotentes Induzidas/fisiologia , Iris/citologia , Regeneração Nervosa/fisiologia , Neurônios Retinianos/fisiologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Recoverina/metabolismo , Reprodutibilidade dos Testes , Células Ganglionares da Retina/metabolismo , Neurônios Retinianos/citologia , Teratoma/patologiaRESUMO
The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.
Assuntos
Cálcio/metabolismo , Recoverina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Dicroísmo Circular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/química , Transferência Ressonante de Energia de Fluorescência , Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Íons/química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Recoverina/química , Recoverina/genéticaRESUMO
Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL-/- ), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL-/- OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL-/- OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Células-Tronco Embrionárias Humanas/metabolismo , Organoides/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Sistemas CRISPR-Cas , Diferenciação Celular , Éxons , Edição de Genes/métodos , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Opsinas/genética , Opsinas/metabolismo , Organoides/patologia , Recoverina/genética , Recoverina/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Receptor X Retinoide gama/genética , Receptor X Retinoide gama/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.