Valproic acid impairs astroglial differentiation of neural stem cells by inhibition of PI3-kinase/Akt/NFkB pathway

Background: VPA is a known inhibitor of PI3-kinase/Akt pathway. Inhibitory effects on GSK-3 (downstream effector of PI3-kinase), as well as direct effects on PI3-kinase are reported. Own previous studies showed that wortmannin (PI3-kinase inhibitor) and SN50 (NFkB-inhibitor) blocked astroglial differentiation of mesencephalic neural stem cells (NSCs). Neural differentiation promoting effects of VPA are as well discussed as effects inhibiting astroglial differentiation. Here we examined the effect of VPA on astroglial, oligodendroglial and neuronal differentiation. Toxic effects of VPA in used dosages were excluded in previous studies.

Methods: NSCs were maintained in DMEM/F-12 (2:1) with 2% B27, 2% penicillin/streptomycin, 20ng/ml EGF (37°C, humidified atmosphere, 5% CO2, 92% N2, 3±2% O2. NSCs were differentiated on poly-L-lysine in P4–8F without or with up to 300µg/ml VPA for up to 10 days. ELISA was carried out using FACE® kit. Immunocytochemistry was carried out using standard protocols. Antibodies: mouse anti-GalC monoclonal 1:300, rabbit anti-GFAP protein 1:750, mouse anti Tuj 1:500 (all Chemicon), mouse anti-MAP2 1:300 (BD Bioscience) and fluorescence labeled secondary antibodies (Invitrogen).

Results: After differentiation in the presence of 300µg/ml VPA the number of GFAP+ cells was reduced to 56.6±3.0% (65.7±1.4% without VPA, p=0.02). For the number of GalC+ cells (11.8±2.30%/12.3±3.8; VPA/no VPA) we found no difference. For the number of Map2+ cells there was a tendency towards more neurons under VPA treatment (66.5±11.5%/51.0±12.1%; VPA/no VPA, p=0.26). VPA treatment led to a marked NFkB downregulation after 1 and 6h (p=0.04, p=0.05), whereas on p38 and JNK kinases, no significant influence could be detected.

Conclusions: Consistent with our hypothesis VPA treatment inhibited astroglial differentiation of fetal mesencephaltic neural stem cells by negatively influencing the PI3-kinase/Akt pathway to NFkB. Theses findings are compatible to previous publications in other cell systems showing the inhibitory effect of VPA on the PI3-kinase/Akt pathway as well as to a previous study of our own group showing that inhibition of the PI3-kinase/Akt pathway blocks astroglial differentiation. Other groups showed that VPA did facilitate neuronal differentiation in different cell systems. This finding could not be statistically reproduced, but there was a non-significant tendency.

Supported by the Forschungsverbund Neurowissenschaften of the EMAU HGW.