Neurotrophin-4 (ntf4) mediates neurogenesis in mouse embryonic neural stem cells through the inhibition of the signal transducer and activator of transcription-3 (stat3) and the modulation of the activity of protein kinase B

Abstract

The effect of neurotrophin-4 (Ntf4) on mouse embryonic (day-14) neural stem cell (mE14-NSC) fate determination and the mechanisms involved were investigated. Using primary mE14-NSCs, immunocytochemistry and molecular-cell biological methods, such as Western-blotting, we characterized the effect of Ntf4 on mE14-NSC differentiation. Obtained in-vitro data revealed an interesting phenomenon of Ntf4 action resulting in enhanced mE14-NSC commitment to progenitor cells of the neuronal lineage. During this process, Ntf4 suppresses the interleukin 6 (Il6) family receptor and the Notch signalling pathways by modulating their specific receptor cleavages. The observed lineage commitment is controlled via an Ntf4-mediated modulation of protein kinase B (PKB/Akt) activity and characterized by a decreased Stat3 (signal transducer and activator of transcription-3) phosphorylation status. These findings suggest that the Ntf4-activated signalling cascade is responsible for initiating a concert among sheddases, kinases, and phosphatases to mediate neurogenesis.

Fig. 1

Ntf4’s dosage effects on mE14-NSCs. a Western blot analysis of mE14-NSCs grown as indicated in the presence of Egf and Ntf4 for three passages. Molecular weight markers are indicated in kDa. As control alpha-tubulin (Tuba1a) is shown for equal loading. Experiments were repeated three times and representative blots are shown. b and c Quantitative analysis of the Western-blots shown in a. *P < 0.05, comparing Egf [5 ng/ml and Ntf4 (0 ng/ml)] with Egf [5 ng/ml plus Ntf4 (100 ng/ml)]

Fig. 2

Ntf4 increases neuronal differentiation in mE14-NSCs. mE14-NSCs were grown (+Egf) in the absence (left) or presence (right) of Ntf4 (100 ng/ml) for three passages before differentiated in NB/B27 for 5 days and ICC was performed with Gfap (green), Tubb3 (red) and DAPI (blue, counterstaining of nuclei). Ntf4-treated cells clearly show a significant higher amount of neurons (red Tubb3⁺ cells). Experiments were repeated three times and representative pictures are shown. (Color figure online)

Fig. 3

Time-dependent inhibition of Stat3 activity in Ntf4-stimulated mE14-NSCs. a Western blot analysis revealed the changes of the phosphorylation levels of Stat3, Mapk1/3, Akt1/3 (~62 kDa) and Akt2 (~55 kDa) over a period of 24 h with a significant early inhibition of Stat3 activity that, however, returns back even beyond basal levels after 24 h. mE14-NSCs were grown and stimulated as described in “Materials and Methods” for the indicated time. Experiments were repeated three times and representative blots are shown. b A graphic representation showing the trend of phosphorylation level alterations of the substrates analysed in a. (Color figure online)

Fig. 4

Proposed Ntf4 signalling during the differentiation of NSCs into neuronal progenitors. Ntf4 activates Erk1/2 (Mapk1/3) and Shp2 resulting (1) in the dephosphorylation and inhibition of Stat3 and (2) the shedding and inhibition of the Il6 family receptors Lifr and gp130 (Il6st), both by substrate sensitization and Tace activation. Concurrently, Ntf4 antagonizes Notch-1 signaling (reduced cleavage) by increasing the shedding of its competitor APP by the γ-secretase. All these Ntf4-initiated effects enhance the permission of NSC commitment towards the neuronal lineage. (Color figure online)