Prpar Mihevc Sonja, Pavlin Mojca, Darovic Simona, Živin Marko, Podbregar Matej, Rogelj Boris, Mars Tomaz
Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia.
Nano and Biotechnological Applications Group, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.
J Mol Neurosci. 2017 Aug;62(3-4):318-328. doi: 10.1007/s12031-017-0940-y. Epub 2017 Jun 15.
Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS. This model was first characterised for endogenous expression and distribution of ALS-related proteins TDP-43 and FUS. Then, wild-type FUS and its mutants were introduced into these co-cultures to determine how FUS defects in nuclear transport modulate the pathological conditions. FUS-bearing plasmids were introduced by classical transfection and electroporation, as novel approaches to deliver plasmids into explants, and their cellular distributions were characterised. Endogenous nuclear expression of TDP-43 and FUS was observed in explants and myoblasts/myotubes. After transfection, wild-type FUS was expressed in nuclei of myoblasts, myotubes and explants, although with low transfection rates. Following successful electrotransfection into explants, the localisation of wild-type FUS was nuclear, and it was detected in neurons, astrocytes, Schwann cells and oligodendrocyte precursors, whereas the FUS∆Y, FUSY526A and FUSY526E mutants were cytoplasmic, and the FUSY526F mutant was nuclear and cytoplasmic. This co-culture model is applicable to the study of neuronal and non-neuronal cell contributions to ALS and other neurodegenerative diseases, and it can be used to investigate drug targets amenable to intervention.
远端轴突和神经肌肉接头的退化是肌萎缩侧索硬化症（ALS）病理学的早期特征，最终导致运动神经元因轴突回缩和肌肉萎缩而丧失。ALS发病机制中运动神经元、肌肉细胞和伴随的神经胶质细胞之间复杂的相互作用需要合适的实验模型。在此，我们基于由胚胎大鼠脊髓外植体中的神经元支配的人肌管定义了一种共培养模型，以研究ALS的病理学和治疗方法。该模型首先针对ALS相关蛋白TDP-43和FUS的内源性表达和分布进行了表征。然后，将野生型FUS及其突变体引入这些共培养物中，以确定核转运中的FUS缺陷如何调节病理状况。通过经典转染和电穿孔引入携带FUS的质粒，作为将质粒导入外植体的新方法，并对其细胞分布进行了表征。在外植体和成肌细胞/肌管中观察到TDP-43和FUS的内源性核表达。转染后，野生型FUS在成肌细胞、肌管和外植体的细胞核中表达，尽管转染率较低。成功电转染到外植体后，野生型FUS的定位是细胞核，并且在神经元、星形胶质细胞、雪旺细胞和少突胶质前体细胞中检测到，而FUS∆Y、FUSY526A和FUSY526E突变体是细胞质的，FUSY526F突变体是细胞核和细胞质的。这种共培养模型适用于研究神经元和非神经元细胞对ALS和其他神经退行性疾病的影响，并且可用于研究适合干预的药物靶点。
