Overexpression of neuronal intermediate filament internexin in the PC-12 cell line

• Main Authors: Tzu-Chiang Liu, 劉自強
• Other Authors: 錢宗良
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/57299301444864791801

碩士 === 國立臺灣大學 === 解剖學暨細胞生物學研究所 === 92 === The neuronal intermediate filaments include not only the neurofilament triplet proteins but also peripherin and internexin. Both peripherin and internexin are expressed in the neurons of PNS and CNS during development. To determine whether neurite outgrowth is enhanced by internexin, the cDNA of rat internexin tagged with EGFP was transfected into a rat adrenal pheochromocytoma cell line PC-12 that responds to nerve growth factor (NGF) by induction of the neuronal phenotype. After G418 selection, 5 stable cell lines were established. Selected stable clones were induced by NGF and examined for expression patterns of neuronal intermediate filaments by western blot and immunocytochemistry. Differentiating neurons were also collected after NGF induction for RT-PCR analyses. Comparing the internexin-EGFP transfected stable cell line with non-transfected PC-12 cells, overexpressed internexin-EGFPs were found mainly in cell bodies and the proximal part of neurites. It was also found that overexpression of internexin-EGFPs enhanced the neurite outgrowth of PC-12 cells at the early stages of NGF induction. NF-L and NF-M were up-regulated by the overexpression of internexin-EGFPs. Interestingly, internexin-EGFP transfected cells detached from culture plates obviously at the later stages of NGF induction. Massive IF accumulations, swelling mitochondria, degenerating neurites with numerous electron-dense granules were observed ultrastructurally in the internexin-EGFP transfected cells. These observations may imply that cell death was undergoing in internexin-EGFP transfected cells. From this study, it could be suggested that internexin may play an important role in the neurite outgrowth and regulate the expression of other neurofilaments during neuronal development. Apoptosis-like cell death could be also induced by the overexpression of internexin-EGFP in PC-12 cells after the NGF induction.