Purpose: Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases, however the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs including exosomes encapsulate and transfer nucleic acids, including microRNA (miRNA), to recipient cells which in disease may result in dysfunctional immune responses and a loss of homeostatic regulation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Methods: Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results: Results demonstrated an inverse correlation between s-mEV secretion and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Conclusions: Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.

中文翻译：

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视网膜健康和变性中的细胞外囊泡及其miRNA转运：体内平衡的介质和靶向基因治疗的载体。

目的：已知在视网膜变性疾病中会逐渐发生光感受器细胞死亡和炎症，但是这些生物学过程的分子机制在很大程度上尚不清楚。细胞外囊泡（EV）是细胞间通讯的重要介体，在调节免疫反应中起着新的作用。包括外来体在内的电动汽车将包括microRNA（miRNA）在内的核酸封装并转移到受体细胞中，这在疾病中可能导致功能失调的免疫反应和体内稳态调节的丧失。在这项工作中，我们调查了孤立的视网膜中小型EV（s-mEV）的作用，其中包括健康和退化性视网膜中的外泌体。方法：使用动态光散射对正常视网膜中分离出的s-mEV进行表征，透射电子显微镜和蛋白质印迹，并使用纳米跟踪分析在5天的光氧化损伤诱导的变性中进行定量。小RNAseq用于表征从健康和受损的视网膜分离出的视网膜s-mEV的miRNA。最后，使用系统性每日施用外泌体抑制剂GW4869和s-mEV丰富的miRNA miR-124-3p的原位杂交来进行外泌体抑制对细胞间miRNA转移和免疫调节的影响。进行了视网膜电图和免疫组织化学分析，以评估由于GW4869诱导的外来体耗竭而导致的视网膜功能和形态变化。结果：结果表明，s-mEV分泌与光感受器生存能力呈负相关，变性后s-mEV数量减少。小RNAseq显示，与整个视网膜组织相比，s-mEVs包含独特富集的miRNA，但是，光氧化损伤后s-mEV miRNAnome没有差异。还发现通过使用GW4869抑制外泌体会加剧视网膜变性，在抑制外泌体的小鼠发生光氧化损伤后，视网膜功能降低，炎症和细胞死亡水平升高。此外，在损伤过程中，用GW4869处理的小鼠显示出源自感光器的miR-124-3p向内视网膜的易位。结论：综上所述，我们认为视网膜s-mEV及其miRNA货物在通过免疫调节维持视网膜稳态方面起着至关重要的作用，并有潜力用于视网膜退行性疾病的靶向基因治疗。