淀粉样蛋白-β(Aβ),一个易于聚集和神经毒性肽家族,与年龄相关性黄斑变性(AMD)的病理生理学有关。我们以前已经表明,Aβ42的寡聚和纤维状物种对大鼠产生视网膜毒性,但是,虽然暴露于淀粉样蛋白的后果与细胞内作用有关,视网膜中Aβ42内化的机制尚未得到很好的表征。在大脑中,67kDa层粘连蛋白受体(67LR)参与Aβ相关神经元细胞死亡。一种源自色素上皮衍生因子(PEDF)的短肽,以前命名为PEDF-335,被发现通过靶向67LR减轻缺血性视网膜病变的实验模型.在本研究中,我们假设67LR介导视网膜中致病性Aβ42集合的摄取,PEDF-335对该受体的靶向可能会限制Aβ的内化,从而改善其视网膜毒性。为了测试这一假设,在用Aβ42的纤维状或寡聚结构处理之前,将培养物中的ARPE-19细胞与PEDF-335孵育。免疫染色证实,PEDF-335处理基本上防止淀粉样蛋白内化进入ARPE-19细胞,并在存在毒性寡聚和纤维状Aβ42实体的情况下在体外维持它们的活力。进行FRET竞争测定并证实PEDF-335与RPE样细胞中的67LR的结合。在向双眼施用视网膜毒性Aβ42低聚物或原纤维之前2天,在实验眼中用玻璃体内PEDF-335处理野生型大鼠。在注射后6周通过视网膜电图评估视网膜功能。在先前用玻璃体内PEDF-335治疗的眼中,用寡聚或纤维状Aβ42组装体治疗的大鼠的ERG反应接近正常,而在单独注射Aβ42组装体治疗的对照眼中测量的那些显示视网膜功能在6周内的病理性衰减。通过免疫染色和蛋白质印迹离体确定67LR的视网膜存在。视网膜染色显示67LR主要在视网膜核层中的结构表达。在Aβ42的存在下,67LR的水平增加,尽管它的视网膜分布基本上没有改变。相比之下,单独暴露于PEDF-335后,67LR的视网膜表达水平没有明显差异,其在视网膜中的定位模式同样主要集中在内部和外部核层。总之,我们发现PEDF-335赋予抗Aβ42介导的视网膜毒性的保护作用,在大鼠体内和细胞中都具有显着的作用。PEDF-335在视网膜中的作用可能通过结合67LR和通过至少部分抑制Aβ42内化来介导。这些结果表明,PEDF-335在视网膜中淀粉样蛋白相关毒性的靶向抑制的发展中可能值得进一步考虑。更广泛地说,我们的观察结果为视网膜中细胞外与细胞内Aβ42的重要性提供了证据,并提出了有关Aβ视网膜致病性分子机制的概念。
Amyloid-beta (Aβ), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aβ42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aβ42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aβ-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aβ42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aβ, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aβ42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aβ42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aβ42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aβ42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aβ42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aβ42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aβ42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aβ42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aβ42 in the retina and suggest concepts on the molecular mechanism of Aβ retinal pathogenicity.