Istituto per la Ricerca e l' Innovazione Biomedica (IRIB) CNR, via U. La Malfa 153, 90146, Palermo, Italy.
Istituto di Biofisica (IBF) (sez. Palermo) CNR, via U. La Malfa, 153, 90146, Palermo, Italy.
J Nanobiotechnology. 2021 Jan 6;19(1):6. doi: 10.1186/s12951-020-00748-6.
Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems.
Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as a vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria.
Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, the replacement of affected mitochondria with healthy ones could be a potential therapy for treating neuronal mitochondrial dysfunction-related diseases.
线粒体功能障碍是神经退行性疾病发病和进展的关键因素。最近，线粒体移植被建议作为一种创新和有吸引力的策略，用于转移和替代受损的线粒体。在这里，我们首次提出使用大鼠脑提取的突触体作为线粒体传递系统，突触体是分离的突触末端的亚细胞部分，其中包含线粒体。
突触体的制备通过突触小体蛋白和 PSD95 的存在得到了验证。突触体的特征在于尺寸、zeta 电位、多分散指数和颗粒/ml 数。尼罗红或 CTX-FITCH 标记的突触体通过涉及特定蛋白质-蛋白质相互作用的机制被内吞到 LAN5 受体细胞中，这一点通过胰蛋白酶处理后融合能力丧失以及使用不同的细胞系得到了证明。通过突触体和 LAN5 细胞中姜黄素的存在证明了突触体的加载和释放能力。通过存在 Opa1、Fis1 和 TOM40 线粒体蛋白以及 JC-1 测量证明了由突触体转移的线粒体的活力。此外，突触体将有活力的线粒体递送到神经元细胞的细胞质中，这通过显微镜图像、TOM40、细胞色素 c、己糖激酶 II 线粒体蛋白的增加以及大鼠线粒体 DNA 的存在得到了证明。最后，通过使用突触体作为载体，健康的线粒体恢复了含有鱼藤酮或 CCCp 损伤线粒体的细胞中的线粒体功能。
综上所述，这些结果表明突触体可以作为将分子和细胞器递送到神经元细胞的天然载体。此外，用健康的线粒体替代受损的线粒体可能是治疗与神经元线粒体功能障碍相关疾病的潜在疗法。
