The multi-phosphorylation repeat of human NF-M examined in human neurons generated from retinoic acid treated NTera-2 cells

Neurofilament proteins are the predominant cytoskeletal proteins of axons. Although these proteins are the most abundant protein in axons there is no consensus as to their functional role in maintaining the structure of axons during development and adulthood. The present study addresses the issue of the regulation of neurofilament phosphorylation by examining the expression and phosphorylation of neurofilament proteins in a novel cell culture system developed for this purpose. The initial studies examined the phosphorylation of human NF-M when expressed by transfection in non-neuronal cells in the hopes that a simplified system would allow an understanding of the regulation of neurofilament phosphorylation. While NF-M was phosphorylated in these cells at the appropriate sites, the regulation and extent of these phosphorylation events was dramatically different than that seen in neurons. A more meaningful way to study the function of any neuronal cytoskeletal proteins is in a neuronal context thus it was necessary to develop a cell culture system suitable for this purpose. This system must consist of cells which highly resemble neurons but which retain certain of the advantageous properties of cell lines. Manipulation of a human teratocarcinoma cell line, NTera 2/D1, following retinoic acid treatment yielded such a system. Using this method it was possible to generate unlimited numbers of post-mitotic cells which closely resemble neurons in all features examined. Success in generating these cultures from this cell line was particularly dependent on the phenotype of these cells. NTera-2/D1 cells have a phenotype resembling neuronal precursor cells before treatment with retinoic acid rather than a pluripotential stem cell phenotype. Using this culture system I examined the regulation of phosphorylation within the multi-phosphorylation repeat of human NF-M