Overexpression of HuD accelerates neurite outgrowth and increases GAP-43 mRNA expression in cortical neurons and retinoic acid-induced embryonic stem cells in vitro.
The neuron-specific RNA-binding protein HuD binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and stabilizes the mRNA. We have previously shown that overexpression of HuD in PC12 cells increases GAP-43 protein expression and induces the spontaneous formation 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 using two in vitro systems: E19 rat cortical neurons and retinoic acid (RA)-induced embryonic stem (ES) cells. Normal neurite outgrowth of cortical neurons 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-deficient HSV-1 vector containing the HuD cDNA in the sense orientation (HSV-HuD). Overexpression of HuD accelerated the formation of neurites. Immunocytochemical analysis showed that excess HuD resulted in a threefold increase in the number of GAP-43-positive cells undergoing morphological differentiation after 24 h of treatment. Using in situ hybridization, we found that the increased HuD expression resulted in a twofold increase in the levels of GAP-43 mRNA. Similarly, overexpression of HuD in RA-induced embryonic stem cells was found to increase the number of GAP-43-positive cells undergoing process 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.
Anderson
K D
KD
Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
Sengupta
J
J
Morin
M
M
Neve
R L
RL
Valenzuela
C F
CF
Perrone-Bizzozero
N I
NI
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D051959:ELAV Proteins; D004622:Embryo, Mammalian; D005260:Female; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D007641:Keratolytic Agents / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D051959:ELAV Proteins; D019922:GAP-43 Protein; D007641:Keratolytic Agents; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014212:Tretinoin