Valproic acid promotes neuronal differentiation by induction of proneural factors in association with H4 acetylation
Introduction
Valproic acid (VPA) (2-propylpentanoic acid) is a well known anticonvulsant and mood stabilizer (Henry, 2003). Recent experiments in 293T, Neuro2A, and teratocarcinoma F9 cells have demonstrated that VPA can directly inhibit histone deacetylase inhibitor (HDAC) activity and cause hyperacetylation of histones (Göttlicher et al., 2001, Phiel et al., 2001). HDACs modify the N-terminal tails of histones and alter their interaction with DNA, thus serving as epigenetic regulators of gene expression. HDAC inhibition mediated by VPA has been shown to suppress the growth and increase the differentiation of many different tumor cell lines (Blaheta and Cinatl, 2002). In the nervous system, VPA stimulates the proliferation of rat neural progenitor cells prepared from the lateral ganglionic eminence or neocortex, and increases neuronal differentiation associated with γ-amino butyric acid (GABA) expression at the expense of astrocytic differentiation (Laeng et al., 2004). However, it has been reported that VPA inhibits proliferation and induces neuronal differentiation of adult hippocampal neural progenitor cells in vitro and in vivo (Hsieh et al., 2004) and that this effect is associated with hyperacetylation of histones and upregulation of the neurogenic basic helix-loop-helix (bHLH) transcription factor NeuroD (Hsieh et al., 2004).
Because the acetylation state of histones modulates chromatin structure and regulates gene expression, we hypothesized that VPA might influence the expression of other proneural and negative transcription factors responsible for neuronal differentiation in vitro and in vivo. In this study we examined whether the acetylation induced by VPA regulates proneural transcriptional factors and cell cycle-related genes, and whether it affects the timing of neuronal differentiation. HDAC inhibition appears to be an important mechanism controlling the onset of gene expression leading to neuronal differentiation in vitro because VPA was found to be required to upregulate several proneural genes and downregulate differentiation inhibitors.
Section snippets
Culture of hippocampal neural progenitor cells
We used adult pregnant Sprague– Dawley rats (Harlan Sprague– Dawley, Indianapolis, IN) and the conditions used by Amoureux et al., 2000, Johe et al., 1996 and Kim et al. (2004) with slight modifications. Briefly, hippocampi were dissected from embryonic day 16.5 (E16.5) embryos into Hank's balanced salt solution (HBSS) without calcium or magnesium. The dissociated cells were plated on 10-cm-diameter dishes coated with 15 μg/ml poly-l-ornithine and 1 μg/ml fibronectin (Invitrogen, Carlsbad, CA) at
VPA decreases proliferation of embryonic hippocampal neural progenitor cells
It has been reported that the short-chain fatty acid type HDAC inhibitor, VPA, reduces proliferation of adult hippocampal neural progenitors in vitro and in vivo (Hsieh et al., 2004). However, Laeng et al. reported that it increased proliferation of neuronal progenitors derived from the embryonic cortex (Laeng et al., 2004). Therefore we tested whether it stimulated the proliferation of neuronal progenitors derived from the embryonic hippocampus. Progenitors at 2 days after passage were treated
Discussion
It is clearly established that HDAC inhibition increases neuronal differentiation in the hippocampus (Phiel et al., 2001, Göttlicher et al., 2001), but relatively little is known about the molecular mechanisms underlying this event. In the present study we used the well-established model system of multipotent neural progenitor cells to investigate the connection between histone acetylation and neuronal gene expression. We provide three novel insights into the functions of VPA in hippocampal
Acknowledgements
This research was supported by a grant (M103KV010008-06K2201-00810) awarded to H. Son from the Brain Research Center (BRC) of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea, the Outstanding Woman Scientist Support Program (2005-KRF-204-E00057) from Korea Research Foundation (KRF) awarded to H. Son, and partially supported by Basic Research Promotion Fund (KRF-2007-314-C00252) from KRF.
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These authors contributed equally to this work.