A proapoptotic effect of valproic acid on progenitors of embryonic stem cell-derived glutamatergic neurons

R Fujiki,A Sato,M Fujitani,T Yamashita

doi:10.1038/cddis.2013.205

R Fujiki, A Sato + Show 2 more

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https://doi.org/10.1038/cddis.2013.205

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Abstract

Valproic acid (VPA) is a branched-chain saturated fatty acid with a long history of clinical use as an antiepileptic drug (AED). VPA is also known to inhibit histone deacetylases (HDACs) and to cause diverse effects on neural progenitor cells (NPCs) and neurons. Although the neuroprotective or neurodestructive effects of VPA have been investigated in heterogeneous cell populations, in this study, we used homogeneous populations of NPCs and glutamatergic cortical pyramidal neurons, which were differentiated from embryonic stem (ES) cells. At therapeutic concentrations, VPA had a proapoptotic effect on ES cell-derived NPCs of glutamatergic neurons, but not on their progeny. This effect of VPA most likely occurred through the inhibition of HDACs, because similar phenotypes were observed following treatment with other HDAC inhibitors (HDACis) such as trichostatin A and sodium butyrate. The proapoptotic phenotype was not observed when cells were exposed to a structural analog of VPA, valpromide (VPM), which has the same antiepileptic effect as VPA, but does not inhibit HDACs. Western blotting confirmed that treatment with HDACis, but not VPM, significantly increased the levels of histone H3 acetylation in NPCs. HDACi treatments did not affect the survival of neurons, although the acetylation levels were increased to a limited extent. These results, which are based on a homogeneous culture system, suggest that VPA inhibits HDAC activity and induces the apoptosis of NPCs that are fated to differentiate into glutamatergic neurons. The dose-dependent effects of VPA both on apoptosis and hyperacetylation of histone H3 in NPCs supported this notion. These cell type- and differentiation stage-specific effects of VPA imply that dysfunction of HDACs during pregnancy significantly increase the risk of congenital malformations associated with VPA administration.

Highlights

(AED); it is widely used for the treatment of mood disorders.[1]
Taken together with previous reports,[25,27] these data suggest that VPA, primarily by the inhibition of histone deacetylases (HDACs), suppresses apoptosis and induces neuronal differentiation of heterogeneous neural progenitor cells (NPCs), but contrarily induces apoptosis of homogeneous NPCs that are fated to differentiate into glutamatergic neurons
NPCs treated with VPA died within 24 h, so we investigated caspase-3 activation using ICC to quantify apoptotic neurons 15 h after VPA treatment (Figure 1b)

Summary

Introduction

(AED); it is widely used for the treatment of mood disorders.[1]. the mechanism of action of VPA is not fully understood, it may reduce neuronal activity in the human brain by blocking sodium and calcium channels and enhancing g-aminobutyric acid (GABA) function by inhibiting GABA transaminase.[2,3] VPA directly inhibits histone deacetylases (HDACs) and relieves HDAC-dependent transcriptional repression by inducing hyperacetylated histones.[4]. Bibel et al.[33,34] recently established a retinoic acid-treated embryoid body-based differentiation protocol that promotes the generation of highly homogeneous glutamatergic cortical pyramidal neurons from embryonic stem (ES) cells The purity of this population is known to reach 90–95%, which is the highest efficiency of differentiation to glutamatergic neurons ever reported.[35,36] our approach led to substantial gains in neuronal survival.[37] VPA did not have a proapoptotic effect on ES cell-derived glutamatergic neurons, but did demonstrate proapoptotic effects on NPCs at therapeutic concentrations (0.3–0.7 mM).[4,11] The effect of VPA was most likely through the inhibition of HDACs, because similar phenotypes were observed following treatment with either of the two other HDACis, trichostatin A (TSA) and sodium butyrate (NaB). We observed that VPA had dose-dependent effects both on apoptosis and hyperacetylation of histone H3 in NPCs, and this strongly indicates a correlation between hyperacetylation of histones and apoptosis

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