Role of Protein Interactions in Retina Development and Function

蛋白质相互作用在视网膜发育和功能中的作用

• 批准号: 10019981
• 负责人: SOFIA P BECERRA
• 金额: $ 81.24万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10019981
• 关键词: Adhesions; Age related macular degeneration; Aging; Angiogenesis Inhibitors; Animal Model; Antioxidants; Apical; Autophagocytosis; BODIPY; Binding; Biological Assay; CD34 gene; CRISPR/Cas technology; Cell Culture Techniques; Cell Death; Cell Extracts; Cell Line; Cell Survival; Cells; Cellular Stress; Chemotaxis; Choroidal Neovascularization; Chronic; Coculture Techniques; Confocal Microscopy; Corneal Neovascularization; Crystallization; Crystallography; Cultured Cells; Deterioration; Development; Diffusion; Dose; Drops; Dyes; Equus caballus; Exposure to; Eye; FASN gene; FRAP1 gene; Family; Fluorescein; Gene Expression; Genes; Human; Hydrolysis; Hyperglycemia; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Vitro; Individual; Inflammasome; Inflammation; Inherited; Injury; Interest Group; Ionophores; Isothiocyanates; Karyotype; Knock-out; Lead; Ligands; Light; Link; Lipase; Lipids; Longevity; MAPK3 gene; Maintenance; Mammals; Measures; Mediating; Membrane; Messenger RNA; Methods; Microglia; Microscope; Molecular; Neoplasm Metastasis; Neuronal Differentiation; Neurons; Neuroprotective Agents; Nonesterified Fatty Acids; Northern Blotting; Organoids; Oxidative Stress; Peptides; Peroxides; Phagocytosis; Phosphodiesterase Inhibitors; Phospholipase; Phospholipids; Photoreceptors; Physiologic pulse; Production; Protein Analysis; Protein Tyrosine Kinase; Proteins; Proto-Oncogenes; Purinoceptor; RNA; Rattus; Recombinant Proteins; Recombinants; Recovery; Research; Residual state; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Neovascularization; Retinal Photoreceptors; Retinal Pigments; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Role; SILV gene; Serpin Superfamily; Shoes; Signal Pathway; Small Interfering RNA; Spectrophotometry; Stains; Stem cells; Structure of retinal pigment epithelium; TREM2 gene; Technology; Time; Tissues; Triglycerides; Tyrosinase related protein-1; Western Blotting; Work; beta-Hydroxybutyrate; cytotoxicity; experimental study; expression vector; fatty acid metabolism; fractalkine receptor; immunoreactivity; induced pluripotent stem cell; interest; melanocyte; member; milligram; neuronal survival; novel; pigment epithelium-derived factor; pigment epithelium-derived factor receptor; pluripotency; postnatal; prevent; protective effect; protective factors; protein biomarkers; receptor; response; retinal ischemia; sodium iodate; stressor; synthetic peptide; tool; transcription factor; transcriptome sequencing; tumorigenesis; vapor; vector; zaprinast

The protective effects of PEDF peptides in retinal cells continued to be investigated. The cone photoreceptor-like cell line, 661W, was used to evaluate in vitro protective effect of PEDF and PEDF derived peptides against experimentally-induced cell death. Cell death was induced by treatments with a phosphodiesterase inhibitor Zaprinast, an ionophore A23187, an oxidative stressor sodium iodate, hyperglycemia injury or by exposure to damaging light (>20,000 lux). Cell viability and death were assessed in treated cells exposed to different concentration of the effectors for different time periods. Dose response was assayed for PEDF and peptide protective activities against sodium iodate exposure. RNA from 661W exposed to sodium iodate and treated with PEDF and 44mer were isolated for Bax and Bcl2 expression determination by RT-PCR and for RNAseq analyses. Protection against cytotoxicity in vitro by chronic sodium iodate-mediated injury on RPE cells was further evaluated with PEDF. This method serves to evaluate protective factors against oxidative stress. Cytotoxicity curves of RPE cells with sodium iodate were generated. The antioxidant PEDF and the 44mer peptide from its neurotrophic region were used to optimize protection against sodium iodate-mediated cytotoxicity. Time course of protection with a given concentration of PEDF and then concentration response for protection at a given incubation time were performed. The survival signaling pathways that prevent oxidative stress by PEDF in RPE were explored, e.g., ERK1/2, mTOR, Akt, Bax, Bcl2 by RT-PCR of RNA and western blotting of protein cell extracts. The survival effects of PEDF in retinal photoreceptors during development in vitro were continued to be investigated. Pure neuronal cultures were prepared from retinas of rats at postnatal day 1, and PEDF-R protein was detected in the cells by western blotting. Polarization of rhodopsin in cells treated with PEDF peptides was evaluated. Human recombinant PEDF protein versions were purified from conditioned media of stably transfected HEK293-Ebna cells with PEDF expression vectors at milligram amounts for crystallization studies. Optimization of production and purification of large amounts of several truncated versions of the human recombinant PEDF-R protein was performed. Quality and recovery of the recombinant proteins was determined by protein analyses, such as SDS-PAGE, western blot, spectrophotometry. PEDFH105A and a synthetic peptide from PEDF-R termed P1 were set for co-crystallization by hanging drop vapor diffusion crystallography. We continued a study to elucidate the role of PEDF-R in the phagocytosis of photoreceptor outer segments (POS). Comparison of phagocytosis activity of RPE cells cultured on transwell membranes vs. plastic was performed. Distribution of PNPLA2 protein, and phagosomal and lysosomal markers was evaluated by immunofluorescence and confocal microscopy in RPE cells undergoing phagocytosis. PNPLA2 mRNA levels following POS addition to the ARPE-19 cell cultures were measured. siRNA technology was used to transiently silence PNPLA2 in the ARPE-19 cell lines and its silencing efficiency was evaluated by RT-PCR and western blot. The mRNA levels of phagocytosis-related genes were assessed in transiently transfected cells with silencing vector following POS addition. We conjugated POS with fluorescein isothiocyanate and used them to assay binding and internalization in control and transiently silenced PNPLA2 ARPE-19 cells. Pulse-chase experiments were also performed and degradation of POS by ARPE-19 cells was followed by free fatty acid production, beta-hydroxybutyrate release, and immunoreactive rhodopsin residual levels. Intracellular neutral lipid accumulation following POS addition in transiently transfected cells was assessed by BODIPY staining under the microscope. The accumulation of peroxidized lipids following POS treatment was assayed using BODIPY C11 dye. An RNA profile of fatty acid metabolism-related genes revealed that PNPLA2 silencing regulated expression of several genes, which lead to further analyses. Comparison of expression of genes of interest (ACAD9, FASN and CPT1C) was further assessed by determining protein levels by western blot. Given that PEDF-R is activated upon its interaction with PEDF, which leads to hydrolysis of phospholipids and triglycerides, the role of the PNPLA2 gene in lipophagy and autophagy in the retina and phagocytosis by RPE begun to be elucidated using tissue derived from human induced pluripotent stem cells (iPSCs). Using gene editing technology, we knocked out PNPLA2 in control iPSC lines using CRISPR-Cas9 technology. Expression of PNPLA2 was evaluated by TaqMan at the mRNA level and absence of protein was confirmed by western blotting. The association of autophagy and inflammasomes is important in aging, in which deterioration in the autophagic capacity and enhanced cellular stress results in inflammasome activation. We used induced pluripotent stem cell (iPSCs) lines from three control individuals and three individuals with age-related macular degeneration. These lines were checked for all pluripotency markers and they had a normal karyotype. Co-cultures of RPE cells and organoids can provide information of the role of autophagy in AMD and how inflammasome is regulated by autophagy. iPSCs were differentiated into RPE cells, and the levels of several specific protein markers were evaluated: Tyrosinase Related Protein 1 (TYRP1) protein levels were evaluated by western blotting, Melanocyte Inducing Transcription Factor (MITF) and premelanosome protein (PMEL17) levels were determined using immunofluorescence staining. In addition, PEDF levels were quantified in media from the apical and basal compartment of trans wells containing iPSC-derived RPE cells to determine their polarization. At the same time, the iPSCs were differentiated into retina organoids/ photoreceptors. Horse shoe eye cups were dissected, and they were kept in ultra-low adhesion plates for another 3 weeks. The presence of pigmented RPE cells and photoreceptors were observed. Co-culture between microglia and retina organoids can provide relevant tools to study the distribution of iMicroglia in the retina and activation of inflammasome in microglia in the presence of oxidative stress. The iPSCs were also differentiated into iPSCs-derived microglia. Hematopoetic stem cells were produced from iPSCs. CD34+ cells were cultured in defined microglia media for 12 days and then they were differentiated into microglia cells. Microglia markers (Purinergic Receptor P2Y12, P2RY12; Triggering receptor expressed on myeloid cells 2, TREM2; receptor for the fractalkine ligand, CXC3CR1; and Proto-oncogene tyrosine-protein kinase MER, Mertk) and their function (phagocytosis, chemotaxis and response to inflammation) were evaluated.

PEDF肽在视网膜细胞中的保护作用仍在继续研究。 锥形感受器样细胞系661W用于评估PEDF和PEDF衍生肽的体外保护作用，以针对实验诱导的细胞死亡。细胞死亡是通过用磷酸二酯酶抑制剂Zaprinast的治疗诱导的，即离子载体A23187，氧化应激剂碘酸钠，高血糖损伤或暴露于损害光（> 20,000 Lux）。 在经历不同时间段的不同浓度的效应子的处理细胞中评估了细胞活力和死亡。分析了针对碘酸钠暴露的PEDF和肽保护活性的剂量反应。暴露于碘酸钠并用PEDF处理的661W和44mer处理的RNA通过RT-PCR和RNASEQ分析分离，用于BAX和BCL2表达测定。 用PEDF进一步评估了碘酸钠介导的RPE细胞损伤对细胞毒性的保护。该方法用于评估防止氧化应激的保护因素。生成了带有碘钠钠的RPE细胞的细胞毒性曲线。来自其神经营养区域的抗氧化剂PEDF和44mer肽用于优化碘酸钠介导的细胞毒性的保护。在给定的PEDF浓度下进行保护，然后在给定的孵育时间进行保护。探索了预防RPE中PEDF氧化应激的存活信号通路，例如，通过RNA的RT-PCR和蛋白质细胞提取物的蛋白质印迹，例如ERK1/2，MTOR，MTOR，AKT，BAX，BCL2。 继续研究了体外发育过程中PEDF在视网膜感受器中的存活效应。 在产后第1天，从大鼠的视网膜中制备了纯神经元培养物，并通过蛋白质印迹在细胞中检测到PEDF-R蛋白。评估了用PEDF肽处理的细胞中视紫红质的极化。 从稳定转染的HEK293-EBNA细胞的条件培养基中纯化了人类重组PEDF蛋白版本，其pEDF表达载体以毫克量进行结晶研究。进行了大量截短版本的人类重组PEDF-R蛋白的优化和纯化。 通过蛋白质分析（例如SDS-PAGE，Western印迹，分光光度法）确定重组蛋白的质量和恢复。通过悬挂液蒸气扩散晶体学来设置PEDFH105A和来自PEDF-R所谓的P1的合成肽进行共结晶。 我们继续进行一项研究，以阐明PEDF-R在光感受器外部段（POS）的吞噬作用中的作用。进行了在Transwell膜上培养的RPE细胞与塑料的吞噬作用的比较。 PNPLA2蛋白的分布以及吞噬和溶酶体标记的分布通过在吞噬吞噬作用的RPE细胞中通过免疫荧光和共聚焦显微镜评估。测量了POS添加为ARPE-19细胞培养物后的PNPLA2 mRNA水平。 siRNA技术用于在ARPE-19细胞系中瞬时沉默PNPLA2，并通过RT-PCR和Western blot评估其沉默效率。添加POS后，用沉默载体在瞬时转染的细胞中评估了吞噬作用相关的基因的mRNA水平。我们将POS与异硫氰酸荧光素共轭，并用它们在对照中测定结合和内在化，并瞬时沉默的PNPLA2 ARPE-19细胞。还进行了脉冲练习实验，然后通过ARPE-19细胞降解POS之后，自由脂肪酸产生，β-羟基丁酸酸酯释放和免疫反应性的Rhodopsin残留水平。通过显微镜下的Bodipy染色评估了瞬时转染细胞中POS添加后的细胞内中性脂质积累。使用Bodipy C11染料测定了POS处理后的过氧化脂质的积累。脂肪酸代谢相关的基因的RNA谱显示PNPLA2沉默调节了几种基因的表达，这导致了进一步的分析。通过蛋白质印迹确定蛋白质水平，进一步评估了感兴趣基因（ACAD9，FASN和CPT1C）表达的比较。 鉴于PEDF-R在与PEDF的相互作用时被激活，这会导致磷脂和甘油三酸酯的水解，因此PNPLA2基因在脂肪噬菌和自噬中的作用在视网膜和自噬中在RPE中通过RPE开始，从而开始使用人类诱导的Pluripotent pluripotent pluripotents sempun（Ipsccccccccccccccccccccc）阐明。使用基因编辑技术，我们使用CRISPR-CAS9技术在控制IPSC线路中淘汰了PNPLA2。通过Taqman在mRNA水平上评估PNPLA2的表达，并通过蛋白质印迹证实了蛋白质的缺失。 自噬和炎症的关联在衰老中很重要，在衰老中，自噬能力恶化并增强了细胞应激会导致炎症体激活。我们使用了来自三个对照个体的诱导多能干细胞（IPSC）线，三个具有与年龄相关的黄斑变性的个体。检查所有多能标记的线条，并具有正常的核型。 RPE细胞和类器官的共培养可以提供自噬在AMD中的作用以及炎性体如何受自噬调节的信息。 IPSC被分化为RPE细胞，评估了几种特定蛋白质标记的水平：通过蛋白质印迹，黑素细胞诱导转录因子（MITF）和前主体蛋白（PMEL17）水平，使用免疫荧光染色来评估酪氨酸酶相关的蛋白1（Tyrp1）蛋白水平。此外，从含有IPSC衍生的RPE细胞的反式井的顶端和基底隔室中对PEDF水平进行了定量，以确定其极化。 同时，IPSC被分化为视网膜器官/感光体。解剖了马鞋眼杯，并将它们保存在超低粘附板中，再保存3周。观察到有色RPE细胞和感光体的存在。小胶质细胞和视网膜器官之间的共培养可以提供相关的工具，以研究在存在氧化应激的情况下在视网膜中分布小胶质细胞中小胶质细胞和炎症体的激活。 IPSC还分化为IPSCS来源的小胶质细胞。由IPSC产生造血干细胞。将CD34+细胞在定义的小胶质细胞培养基中培养12天，然后分化为小胶质细胞。小胶质细胞标记（嘌呤能受体P2Y12，P2RY12；在髓样细胞2，Trem2； trem2； fractalkine配体的受体，CXC3CR1；原始癌基因酪氨酸 - 蛋白 - 蛋白激酶MER，MERTK，MERTK）及其功能（评估了吞噬症和化学响应），对化学症和响应。