Fig 1.
GBE increased the neurite outgrowth of neuroblastoma cells after 3 days of treatment in a 2D cell culture.
Pictures were taken using a confocal microscope (x10). Immunostaining (IMS) with βIII- tubuline/Alexa488: pictures display neurite extension between the cells (upper panels; S1 Fig). Quantification of the neurite outgrowth parameters such as the attachment points (middle panels) and the endpoint numbers (lower panels), after NGF or GBE treatment are shown (Blue: soma, red: neurite, green point: attachment point, yellow point: endpoint).
Fig 2.
GBE increased the neurite outgrowth of neuroblastoma cells after 3 days of treatment in a 2D cell culture.
Quantification of Fig 1 using NeurophologyJ (S1 Table). At 100 μg/ml GBE significantly increased: (A) cell number (soma count), (B) number of neurites (neurite count), (C) neurite length, (D) number of attachment points, (E) number of endpoints, and (F) extent of neurite branching. The effect of GBE was similar than the positive control NGF when compared to the untreated cells. The red line represents the effects of NGF-treated cells. Values represent the mean ±SEM of three independent experiments and were normalized to 100% of untreated CTRL cells. One way ANOVA and post hoc Dunnett’s multiple comparisons versus untreated neuroblastoma cells (CTRL) *P<0.05,**P<0.01,***P<0.001.
Fig 3.
GBE (100 μg/ml) induced neurite extension in a 3D-matrix by increasing the neurite outgrowth of neuroblastoma cells after 3 days of treatment.
Pictures were obtained by merging 3–4 layers of cells (z-stack projection) on 3D-matrix using a confocal microscope (x10). Immunostaining (IMS) with βIII- tubuline/Alexa488: pictures display neurite extension between the cells (upper panels, S2 Fig). Quantification of the neurite outgrowth parameters such as the attachment points (middle panels) and the endpoint numbers (lower panels), after NGF or GBE treatment are shown (Blue: soma, red: neurite, green point: attachment point, yellow point: endpoint).
Fig 4.
GBE (100 μg/ml) increased the neurite outgrowth of neuroblastoma cells after 3 days of treatment using a 3D gel.
Quantification of Fig 3 using NeurophologyJ (S2 Table). GBE (100 μg/ml) ameliorated: (A) cell number (soma count), (B) number of neurites (neurite count), (C) neurite length, (D) number of attachment points, (E) number of endpoints, and (F) extent of neurite branching. The effect of GBE was similar than the positive control NGF when compared to the untreated cells. The red line represents the effects of NGF-treated cells. Values represent the mean ±SEM of three independent experiments and were normalized to 100% of untreated CTRL cells. One way ANOVA and post hoc Dunnett’s multiple comparisons versus untreated neuroblastoma cells (CTRL) *P<0.05. Student t-test +P<0.05,++P<0.01.
Fig 5.
Activation of the Akt/mTOR signaling pathway after GBE (100 μg/ml) treatment in neuroblastoma cells.
Neuroblastoma cells (SH-SY5Y) stimulated with GBE (100 μg/ml) (3 days) were lysed in MILLIPLEX® MAP lysis buffer containing protease inhibitors. 20 μg total protein of each lysates diluted in MILLIPLEX® MAP assay buffer 2 were analyzed according the assay protocol (lysate incubation at 4°C overnight). The Median Fluorescence Intensity (MFI) was measured measurements for the phosphoproteins Akt (Ser473), GSK3β (Ser9), IGF1R (Tyr1135/Tyr1136), mTOR (Ser2448), TSC2 (Ser939) and PTEN (Ser380) with the Luminex® system (S3 Table). The figure represents the average and standard deviation of three replicates. Student t-test *P<0.05, ***P<0.001.
Fig 6.
Possible pathways involved in GBE-induced neuroplasticity pathway.
We propose that neuroprotective effects of GBE are mediated via multiple pathways. In the present study, we show that GBE increases IGF1R phosphorylation (Tyr1135/Tyr1136), which activates the Akt/mTOR pathway. Activation of Akt by phosphorylation (Ser473) inhibits TSC2 (phosphorylation at Ser939) which activates mTOR (phosphorylation at Ser2448) and induces neuronal survival, neurite outgrowth and neuroplasticity. In parallel, GBE inactivates the antagonizing pathways by increasing the phosphorylation of PTEN and GSK3β (at Ser380 and Ser9 respectively). Of note, the growth factors NGF and BDNF activate also the Akt/mTOR pathway, suggesting that GBE may also act via these growth factors to induce its neuroprotective effects. Besides, our previous study showed that GBE increases mitochondrial bioenergetics and decreases reactive oxygen species production (Rhein et al., PlosONE 2009), which may also play a role in neuroprotection and neurite outgrowth. Akt is also known as protein kinase B (PKB). IGF-1R, insulin-like growth factor-1; PI3K, phosphoinositide 3-kinase; PIP2 or PIP3, phosphatidylinositol triphosphate; PTEN, phosphatase and tensin homolog; GSK3, glycogen synthase kinase 3; TSC, tuberous sclerosis protein; mTOR, mammalian target of rapamycin.