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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Mol Psychiatry. 2014 Jan 28;20(2):215–223. doi: 10.1038/mp.2013.192

Figure 5. Akt-1 activation and FGF-21 upregulation are required for the neuroprotective effects of FGF-21 and lithium-VPA co-treatment.

Figure 5

(a) CGCs were transduced with sh-cont or sh-Akt-1 at the time of cell plating, and harvested for FGF-21 mRNA levels at DIV-6 (n=3, two tailed unpaired t-test). (b) CGCs were transduced with sh-Akt-1 as described above and then treated with Li-VPA at DIV-6 and harvested at DIV-8 for FGF-21 mRNA detection using q-PCR analysis (n=4, two-tailed unpaired t-test). (c) CGCs at DIV-6 were treated with 5 nM FGF-21 recombinant protein, or a combination of 3 mM Li and 0.8 mM VPA, followed by exposure to 100 μM glutamate at DIV-12 for 24 hours. Cell viability was determined by MTT assay at DIV-13 (n=3, one-way ANOVA). (d) CGCs were transduced with sh-cont or sh-FGF-21 construct at the time of cell plating, and co-treated with 3 mM Li and 0.8 mM VPA at DIV-6, then harvested for q-PCR assay of FGF-21 mRNA levels at DIV-8 (n=4, two-tailed unpaired t-test). (e) CGCs transduced with sh-cont or sh-FGF-21 as described were co-treated with 3 mM Li and 0.8 mM VPA starting from DIV-6, followed by Western blotting analysis (n=3, one-way ANOVA). (f) At DIV-6, CGCs were treated with 3 mM Li, 0.8 mM VPA, or their combination for 6 days followed by exposure to glutamate for 24 hours. Cell viability was determined at DIV-13 using MTT assay (n=3, two-tailed unpaired t-test). (g) FGF-21 was selectively and robustly induced in aging primary brain neurons by lithium-VPA co-treatment through inhibition of GSK-3 and HDACs, respectively. Lithium and VPA co-treatment induces long-lasting activation of Akt-1 via increased Ser 473 phosphorylation, and this Akt-1 activation is involved in FGF-21 gene expression. Lithium alone only transiently activates Akt-1 and modestly elevates FGF-21 mRNA levels, while VPA alone is ineffective. FGF-21 completely protects primary neurons from glutamate induced excitotoxicity and apoptosis, and these protective effects in return require FGF-21-induced Akt-1 activation. Thus, Akt-1 serves as a feed-forth regulator for the upstream and downstream actions of FGF-21. FGF-21 induction contributes, at least in part, to the synergistic neuroprotective effects of lithium-VPA co-treatment in aging neurons. Quantified data are means ± SEM; *P<0.05; **P<0.01; ***P<0.001.