The neuronal intermediate filaments include not only the neurofilament triplet proteins but also peripherin and α-internexin. To determine whether neurite outgrowth is enhanced by ́-internexin, the cDNA of rat α-internexin tagged with enhanced green fluorescent protein (EGFP) was transfected into a rat adrenal pheochromocytoma cell line PC12 that responds to nerve growth factor (NGF) by induction of the neuronal phenotype. 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 analysis. Overexpressed α-internexin-EGFPs were found mainly in cell bodies and the proximal part of neurites. It was also found that overexpression of α-internexin-EGFPs enhanced the neurite outgrowth of PC12 cells at the early stages of NGF induction. Meantime, NF-L and NF-M were upregulated by the overexpression of α-internexin-EGFPs. Interestingly, α-internexin- EGFP-transfected cells obviously detached from culture plates at the later stages of NGF induction. Massive IF accumulations, swelling mitochondria, and degenerating neurites with numerous electron-dense granules were observed ultrastructurally in the α-internexin-EGFP-transfected cells. In addition, neuronal death was also characterized positively by the TUNEL assay. These observations may imply that cell death was occurring in α-internexin-EGFP- transfected cells. From this study, it could be suggested that α-internexin plays an important role in neurite outgrowth and regulates the expression of other neurofilaments during neuronal development. Apoptosis-like cell death could also be induced by the overexpression of α-internexin-EGFP in PC12 cells after NGF induction.
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