LncRNA NR_030777/ DRP1参与的细胞间线粒体传递在纳米钴致神经元损害中的作用及机制

Intracellular mitochondrial transfer is newly discovered as one of the cross-talk phenomena between cells. Our previous study demonstrated that cobalt nanoparticle can cause significant damage in the brain of rats, neural cells and mitochondria, accompanied by transferring mitochondria from astrocytes to damaged neurons. Mitochondrial transfer related protein DRP1 was downregulated by LncRNA NR_030777. Therefore, we propose that under the regulation of NR_030777/DRP1, astrocytes protect neurons against cobalt nanoparticle by transferring mitochondria to neurons. We intend to use neural cell lines, mice cortex primary culture and assess neural and mitochondrial toxicity in response to CoNPs; label CoNPs pretreated neurons with fluorescent probe, followed by coculture with DsRedMito transfected astrocytes, then apply electron and confocal microscopy to confirm the existence of intracellular mitochondria transfer; pre-stain mitochondria of astrocytes with molecular probe, and use flow cytometry sorting to filtrate neurons in the coculture, in order to determine the protective effects of mitochondrial transfer; unveil the role of LncRNA NR_030777 and DRP1 in mitochondrial transfer by tailoring the gene expression experiments of the two, individually. This study provides new aspects of cross-talks between cells to toxicology field and is of great importance on epigenetic mechanisms of mitochondrial transfer.

胞间线粒体传递是最新发现的细胞间交流现象。我们前期发现，纳米钴可致大鼠脑组织、神经细胞及线粒体损害，伴有星形胶质细胞胞外线粒体向受损神经元传递现象，其相关蛋白DRP1被LncRNA NR_030777负调控。为此，我们提出假说：在NR_030777/DRP1的调控下，星形胶质细胞将线粒体传递给神经元，抵御纳米钴致神经元损害作用。本项目拟用神经细胞系及小鼠皮层原代神经细胞评价纳米钴致神经元及线粒体的损害作用；纳米钴预处理的神经元用荧光探针标记后，与DsRedMito转染的星形胶质细胞共培养，用电镜、共聚焦显微镜证明纳米钴暴露下线粒体传递的存在性；以分子探针预染线粒体，流式细胞分选技术筛选共培养的神经元，确定线粒体传递抵御纳米钴损害的保护作用；利用DRP1及NR_030777的基因表达调控实验，揭示其参与传递的机制；为毒理学细胞间交流研究提供新思路，对线粒体传递的表观遗传机制研究具有重要意义。

细胞间线粒体传递是最新发现的细胞间交流现象。我们前期发现，纳米钴可致大鼠脑组织、神经细胞及线粒体损害，伴有星形胶质细胞胞外线粒体向受损神经元传递现象，其相关蛋白DRP1可被长链非编码RNA NR_030777调控。然而二者在纳米钴处理下线粒体传递中的发挥作用及机制均不明确，未见报道。按照计划，本项目利用荧光特异性标记、敲低/过表达、扫描透射电镜、高内涵、共聚焦显微技术等分子生物学技术，顺利完成以下内容：（1）在大、小鼠和体外人源、小鼠源神经细胞等多层次揭示了纳米钴的神经毒性及线粒体毒效应；（2）首次揭示了在纳米钴致神经元损害背景下，星形胶质细胞线粒体可通过隧道纳米管形式传递致神经元，并帮助后者抵御纳米钴所致神经损害效应；（3）明确了LncRNA NR_030777和DRP1在线粒体传递及隧道纳米管形成中的作用。除项目计划内容，增加完成以下内容：（1）探究了纳米钴处理背景下，PI3K/AKT在隧道纳米管形成中的作用；（2）揭示了纳米钴对秀丽线虫的神经毒性和线粒体损害效应及机制。本研究独辟蹊径地从线粒体传递及细胞间交流角度，探讨了环境重金属纳米颗粒致神经损害中的作用及机制，为预防和治疗常见的环境神经毒物所致的神经毒性提供新的思路和途径，也将为利用特定lncRNA作为环境神经毒物中毒及研发药物的靶点提供坚实的理论依据。