Rodriguez-Losada Noela, Wendelbob Rune, Ocaña M Carmen, Casares Amelia Diaz, Guzman de Villoría Roberto, Aguirre Gomez Jose A, Arraez Miguel A, Gonzalez-Alegre Pedro, Medina Miguel A, Arenas Ernest, Narvaez Jose A
Department Human Physiology, Faculty of Medicine, Biomedicine Research Institute of Malaga (IBIMA C07), University of Malaga, Malaga, Spain.
Department of Didactic Science Education, Faculty of Science Education, University of Malaga, Malaga, Spain.
Front Neurosci. 2020 Dec 21;14:570409. doi: 10.3389/fnins.2020.570409. eCollection 2020.
Emerging scaffold structures made of carbon nanomaterials, such as graphene oxide (GO) have shown efficient bioconjugation with common biomolecules. Previous studies described that GO promotes the differentiation of neural stem cells and may be useful for neural regeneration. In this study, we examined the capacity of GO, full reduced (FRGO), and partially reduced (PRGO) powder and film to support survival, proliferation, differentiation, maturation, and bioenergetic function of a dopaminergic (DA) cell line derived from the mouse substantia nigra (SN4741). Our results show that the morphology of the film and the species of graphene (GO, PRGO, or FRGO) influences the behavior and function of these neurons. In general, we found better biocompatibility of the film species than that of the powder. Analysis of cell viability and cytotoxicity showed good cell survival, a lack of cell death in all GO forms and its derivatives, a decreased proliferation, and increased differentiation over time. Neuronal maturation of SN4741 in all GO forms, and its derivatives were assessed by increased protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), the glutamate inward rectifying potassium channel 2 (GIRK2), and of synaptic proteins, such as synaptobrevin and synaptophysin. Notably, PRGO-film increased the levels of Tuj1 and the expression of transcription factors specific for midbrain DA neurons, such as Pitx3, Lmx1a, and Lmx1b. Bioenergetics and mitochondrial dysfunction were evaluated by measuring oxygen consumption modified by distinct GO species and were different between powder and film for the same GO species. Our results indicate that PRGO-film was the best GO species at maintaining mitochondrial function compared to control. Finally, different GO forms, and particularly PRGO-film was also found to prevent the loss of DA cells and the decrease of the α-synuclein (α-syn) in a molecular environment where oxidative stress has been induced to model Parkinson's disease. In conclusion, PRGO-film is the most efficient graphene species at promoting DA differentiation and preventing DA cell loss, thus becoming a suitable scaffold to test new drugs or develop constructs for Parkinson's disease cell replacement therapy.
由碳纳米材料制成的新兴支架结构，如氧化石墨烯（GO），已显示出与常见生物分子的高效生物共轭作用。先前的研究表明，GO能促进神经干细胞的分化，可能对神经再生有用。在本研究中，我们检测了完全还原的（FRGO）、部分还原的（PRGO）氧化石墨烯粉末和薄膜支持源自小鼠黑质的多巴胺能（DA）细胞系（SN4741）存活、增殖、分化、成熟及生物能量功能的能力。我们的结果表明，薄膜的形态以及石墨烯的种类（GO、PRGO或FRGO）会影响这些神经元的行为和功能。总体而言，我们发现薄膜种类的生物相容性优于粉末。细胞活力和细胞毒性分析显示细胞存活良好，所有GO形式及其衍生物均无细胞死亡，随着时间推移增殖减少而分化增加。通过酪氨酸羟化酶（TH）、多巴胺转运体（DAT）、谷氨酸内向整流钾通道2（GIRK2）以及突触蛋白（如突触小泡蛋白和突触素）的蛋白质水平升高，评估了所有GO形式及其衍生物中SN4741的神经元成熟情况。值得注意的是，PRGO薄膜增加了Tuj1的水平以及中脑DA神经元特异性转录因子（如Pitx3、Lmx1a和Lmx1b）的表达。通过测量不同GO种类修饰的氧消耗来评估生物能量学和线粒体功能障碍，对于相同的GO种类，粉末和薄膜之间存在差异。我们的结果表明，与对照相比，PRGO薄膜在维持线粒体功能方面是最佳的GO种类。最后，在诱导氧化应激以模拟帕金森病的分子环境中，还发现不同的GO形式，特别是PRGO薄膜能防止DA细胞丢失和α-突触核蛋白（α-syn）减少。总之，PRGO薄膜是促进DA分化和防止DA细胞丢失最有效的石墨烯种类，因此成为测试新药或开发帕金森病细胞替代治疗构建体的合适支架。
