Overexpression of HuD accelerates neurite outgrowth and increases GAP-43 mRNA expression in cortical neurons and retinoic acid-induced embryonic stemcells in vitro

The neuron-specific RNA-binding protein HuD binds to a U-rich regulatory el ement of the 3 ' untranslated region (3 ' UTR) of the GrAP-43 mRNA and stab ilizes the mRNA, We have previously shown that overexpression of HuD in PC1 2 cells increases GAP-43 protein expression and induces the spontaneous for mation of multiple neurites (K, D, Anderson et al, 2000, J, Neurochem, 75: 1103-1114). In this study, we examined the effects of HuD overexpression on the initial stages of neurite outgrowth and on GAP-43 gene expression usin g two in vitro systems: E19 rat cortical neurons and retinoic acid (RA)-ind uced embryonic stem (ES) cells. Normal neurite outgrowth of cortical neuron s in vitro occurs over a 3-day period with a concomitant increase in GAP-43 and HuD expression. Cortical cells were infected with a replication-defici ent HSV-1 vector containing the HuD cDNA in the sense orientation (HSV-HuD) . Overexpression of HuD accelerated the formation of neurites, Immunocytoch emical analysis showed that excess HuD resulted in a threefold increase in the number of GAP-43-positive cells undergoing morphological differentiatio n after 24 h of treatment. Using in situ hybridization, we found that the i ncreased HuD expression resulted in a twofold increase in the levels of GAP -43 mRNA Similarly, overexpression of HuD in RA-induced embryonic stem cell s was found to increase the number of GAP-43-positive cells undergoing proc ess outgrowth. In conclusion, our results demonstrate that HuD functions in the initiation of neurite outgrowth in a manner due, at least in part, to its regulation of GAP-43 expression. (C) 2001 Academic Press.