Wakx Anaïs, Dutot Mélody, Massicot France, Mascarelli Frédéric, Limb G Astrid, Rat Patrice
Appl Biochem Biotechnol. 2016 Jan;178(2):368-81. doi: 10.1007/s12010-015-1878-6.
Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-β peptide (Aβ); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aβ toxicity on Müller cells? Retinal Müller glial cells were incubated with Aβ for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aβ incubation. For the first time, we showed that Aβ induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aβ cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aβ toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.
视网膜穆勒神经胶质细胞已被认为与年龄相关性黄斑变性（AMD）有关。AMD的特征是有毒的淀粉样β肽（Aβ）积累；我们提出的问题如下：已知在几种退行性疾病中起重要作用的P2X7受体是否参与Aβ对穆勒细胞的毒性作用？将视网膜穆勒神经胶质细胞与Aβ孵育48小时。使用alamarBlue测定法评估细胞活力，使用乳酸脱氢酶（LDH）释放测定法评估细胞毒性。通过共聚焦显微镜观察的免疫标记突出显示P2X7受体表达，并通过YO-PRO-1测定法评估其激活。使用Hoechst 33342评估染色质浓缩，并使用AMC测定法评估半胱天冬酶8和3的激活。在与Aβ孵育之前，将年龄相关性眼病研究2中使用的富含二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）的脂质制剂在细胞上孵育15分钟。首次，我们表明在我们的视网膜模型中，Aβ通过P2X7受体激活诱导非半胱天冬酶依赖性凋亡。DHA和EPA是食品补充剂中推荐用于预防AMD的多不饱和脂肪酸。因此，我们使用富含DHA和EPA的脂质制剂调节Aβ细胞毒性，以更好地理解在临床研究中观察到的结果。我们表明，富含EPA和DHA的鱼油与有效的P2X7受体拮抗剂联合使用，是Aβ毒性的有效调节剂，并且P2X7可能是预防AMD的一个有意义的治疗靶点。
