Functional analysis of rat cerebellum-derived neural differentiation factor

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It is important to find out unknown neural differentiation factors for better understanding of complex central nervous system. We constructed the rat cerebellar cDNA expression library using pCD-2 vector to isolate novel neural differentiation factors. Each cDNA clone in the library was transiently expressed in COS-1/7 cells and the resulting conditioned medium was assayed for the neural differentiation inducing activity against PC12 cells, which are differentiated into sympathetic neuron-like cells in the presence of NGF, cAMP and IL-6. As a result, we isolated cDNA clone 398 (grp75R3), which encodes 67 amino acid residues having high homology to the C-terminal region of rat grp75, one of the heat shock proteins. For the detailed functional analysis of grp75R3, we prepared anti-grp75R3 antibody using GST-fused grp75R3 protein, and also established COS75R3 cells stably expressing grp75R3 protein. The conditioned medium from COS75R3 cells contained a highly active neural differentiation factor when assayed in PC12 cells, however grp75R3 protein was not detected by Western blot with anti-grp75R3 antibody, showing that grp75R3 did not directly differentiate PCl2 cells. By the expriment using antibodies against NGF family neurotrophins such as NGF, BDNF and NT-3 and RT-PCR analysis, it has been shown that the gene expression of NGF was selectively enhnced by grp 75R3 as well as grp75. The same result was obtained when grp75R3 cDNA expression vector was introduced into rat glioma C6 cells. Taking into these data and the significant induction of grp75 expression in the brain of rat starved for 24 hr, it is highly possible that grp75R3/grp75 in glial cells is induced by stress and maintains the survival of NGF-responsive cholinergic neurons.