Bergamin Giorgia, Cieri Domenico, Vazza Giovanni, Argenton Francesco, Mostacciuolo Maria Luisa
Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, 35121 Padova (PD), Italy.
Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, 35121 Padova (PD), Italy.
Biochim Biophys Acta. 2016 Jun;1860(6):1247-55. doi: 10.1016/j.bbagen.2016.03.007. Epub 2016 Mar 9.
Deregulation of axonal transport in neurons is emerging as the major cause of many neurodegenerative diseases in human, such as Charcot-Marie-Tooth (CMT) neuropathy. However, little is known about how mitochondria move in vivo and whether cell culture systems truly represent what happens in living animals. Here we describe the generation of a new zebrafish transgenic line that specifically allows to study mitochondrial dynamics in motor neurons and its application to analyse mitochondrial movement in zebrafish models expressing CMT2A causing mutations.
The Tol2 transposon system was used to generate a transgenic zebrafish line expressing the photoconvertible fluorescent protein Kaede in mitochondria of motor neurons. Mitochondrial shape and movement were monitored by time-lapse confocal live imaging and measured by kymograph analysis. The effects of two well-known CMT causing mutations, L76P and R94Q substitutions in MFN2, were then investigated with the same methods.
We generated the transgenic zebrafish Tg(hb9:MTS-Kaede) line with genetically labelled mitochondria in motor neurons. Kaede protein was correctly and stably targeted to mitochondrial matrix while retaining its photoconvertibility, thus qualifying this model for in vivo studies. Expression of the L76P and R94Q mutations reduced mitochondrial movement in axons and altered mitochondrial distribution in distinct ways.
These findings confirm previously published data obtained in cell cultures and strengthen the hypothesis of different mechanism of action of the two MFN2 mutations. Considering the number of neurodegenerative diseases associated to mitochondrial dynamics, the Tg(hb9:MTS-Kaede) zebrafish line is a promising model to study in vivo alterations of mitochondrial transport underlying human diseases.
神经元轴突运输失调正成为人类许多神经退行性疾病的主要病因，如夏科 - 马里 - 图斯（CMT）神经病。然而，对于线粒体在体内如何移动以及细胞培养系统是否真的能代表活体动物体内发生的情况，我们知之甚少。在此，我们描述了一种新的斑马鱼转基因品系的构建，该品系专门用于研究运动神经元中的线粒体动力学，并将其应用于分析表达导致CMT2A致病突变的斑马鱼模型中的线粒体移动。
利用Tol2转座子系统构建了一个转基因斑马鱼品系，该品系在运动神经元的线粒体中表达可光转换的荧光蛋白Kaede。通过延时共聚焦实时成像监测线粒体的形态和移动，并通过线扫描分析进行测量。然后用同样的方法研究了两种著名的导致CMT的突变，即MFN2中的L76P和R94Q替换的影响。
我们构建了转基因斑马鱼Tg(hb9:MTS-Kaede)品系，其运动神经元中的线粒体带有遗传标记。Kaede蛋白正确且稳定地靶向线粒体基质，同时保留其光转换能力，因此该模型适用于体内研究。L76P和R94Q突变的表达以不同方式减少了轴突中线粒体的移动并改变了线粒体分布。
这些发现证实了先前在细胞培养中获得的数据，并强化了两种MFN2突变作用机制不同的假说。考虑到与线粒体动力学相关的神经退行性疾病的数量，Tg(hb9:MTS-Kaede)斑马鱼品系是研究人类疾病中线粒体运输体内改变的一个有前景的模型。
