Figure 1.
The receptors for SCF and G-CSF are expressed in primary cortical neurons and the growth cones.
Cortical neurons were cultured for 24h and then processed for immunocytochemistry. c-kit (red), receptor for SCF. GCSFR (red), receptor for G-CSF. TuJ1 (green), the signature protein of neurons. F-actin (green), the marker of growth cones. The confocal images on the right columns are the high magnification images of the red boxes inserted in the adjacent panels. Note that c-kit and GCSFR are overlapped with TuJ1-positive neurons. Both c-kit and GCSFR are expressed in the neuron soma and neurites. Moreover, c-kit and GCSFR are also co-localized with F-actin in the neurite growth cones.
Figure 2.
SCF+G-CSF promotes neurite outgrowth.
(A) Measurements of the neurite length 24h after seeding. Note that the neurite length is significantly increased by SCF+G-CSF. (B) SCF+G-CSF promotes neurite extension 48h after plating. Neurite outgrowth was determined with a Neurite Outgrowth Quantification Assay Kit. (C-E) Neurite extension and neuronal network density are enhanced by SCF+G-CSF. Cortical neurons were cultured on transwell membranes with or without SCF+G-CSF treatment for 48h. Neurite outgrowth and neurite network density were examined with immunofluorescent staining on the underside membranes of transwells. (C and D) Immunofluorescent images. (E) Quantification of neurite extension and neuronal network density. (F) Dose-response data. Note that SCF+G-CSF induces neurite outgrowth in a dose-dependent manner. (G-K) Synergy assay data. Cortical neurons were plated on transwell membranes with medium alone (control), G-CSF, SCF or SCF+G-CSF treatment (20ng/ml) for 72h. (G-J) Confocal images of the neurites on the underside membranes of transwells by immunocytochemical staining. (K) Quantification data of synergy assay. Note that SCF alone or in combination with G-CSF (SCF+G-CSF) significantly increases neurite extension, and that SCF+G-CSF shows synergistic effects on the enhancement of neurite outgrowth. *p<0.05, **p<0.01. Mean ± SE; n=3-4. SCF or G-CSF, 20ng/ml unless otherwise noted.
Figure 3.
The involvement of PI3K/AKT signaling in SCF+G-CSF-induced neurite outgrowth.
(A) Western Blotting data. Upper panel (bands): representative bands of phosphorylated AKT (p-AKT) and total AKT (AKT). Lower panel (bar graph): semi-quantitative of the phosphorylated AKT. Note that SCF+G-CSF causes phosphorylation of AKT in a time-dependent manner. Cortical neurons were cultured for 24h and treated with or without SCF+G-CSF, and protein extracts of neurons were collected at selected time to determine phosphorylated AKT. (B) SCF+G-CSF-induced neurite outgrowth is blocked by PI3K signaling inhibitor, LY294002 (LY). Cortical neurons were seeded onto the membranes of transwells for 24h and treated with medium alone (Control), SCF+G-CSF, or SCF+G-CSF+ LY294002 for two days before immunocytochemistry. PI3K inhibitor, LY294002 (20µm), was added 1h prior to SCF+G-CSF treatment. Con: control medium, S+G: SCF+G-CSF, S+G+LY: SCF+G-CSF+LY294002. *p<0.05, **p<0.001. Mean ± SE. N=3.
Figure 4.
NFκB activation and transcriptional activity are enhanced by SCF+G-CSF.
(A) NFκB activation assay data. NFκB activation was determined by IκBα phosphorylation with a FunctionELISA IκBα kit for the quantification of phosphorylated IκBα. Note that SCF and G-CSF alone or in combination induces a significant elevation of phosphorylated IκBα in a time-dependent manner. Markedly, the level of phosphorylated IκBα is significantly increased in SCF+G-CSF-treated neurons at 15 and 30 min after treatment as compared to SCF or G-CSF alone treatment. **p < 0.01: 0 min vs. 15 min, 0 min vs. 30min. ♦♦p < 0.01: SCF+G-CSF vs. SCF, SCF+G-CSF vs. G-CSF at 15 and 30 min after treatment. Mean ± SE, n=6. (B) Dose dependent assay of NFκB transcriptional activity. Quantification of NFκB binding activity was performed using a NFκB Transcription Factor ELISA Kit (p65). Note that both 10 ng/ml of SCF+G-CSF and 50 ng/ml of SCF+G-CSF enhance NFκB binding 1, 3 and 6 h after treatment in comparison with medium control (** p < 0.01). In addition, NFκB binding activity in the treatment of 50 ng/ml of SCF+G-CSF is greater than those of 10 ng/ml of SCF+G-CSF at 1, 3 and 6 h after treatment (** p < 0.01). In the dose of 10 ng/ml, NFκB binding activity is significantly increased 1h after treatment as compared to 3h (# p < 0.05) or 6h (# # p < 0.01) post-treatment. In the dose of 50 ng/ml, a significantly greater NFκB binding activity is seen 1h after treatment than 6h post-treatment (# p < 0.05). Mean ± SE, n=3. (C) NFκB binding activity is changed by SCF and G-CSF alone or in combination treatment. Note that SCF, G-CSF, and SCF+G-CSF cause significantly increases in NFκB binding activity 1h after treatment as compared to the controls (** p < 0.01). However, only SCF+G-CSF shows a long-lasting enhancement of NFκB binding activity as this treatment induces a significant elevation of NFκB binding activity 3 and 6 h after treatment when compared to SCF or G-CSF alone treatment (♦♦p < 0.01). SCF also displays a higher level of activated NFκB 3h post-treatment than the controls (* p < 0.05), whereas there is no difference between SCF and the controls at 6h after treatment. Mean ± SE, n=3. (D) NFκB specific binding assay data. Note that SCF+G-CSF-induced the long-lasting enhancement of NFκB binding activity is completely blocked by wild-type (Wt) NFκB oligonucleotide, whereas a mutant NFκB oligonucleotide (Mut NFκB) fails to prevent the SCF+G-CSF-induced the long-lasting enhancement of NFκB binding activity. Wt: wild-type NFκB oligonucleotide. Mut: mutant NFκB oligonucleotide. ** p < 0.01, * p < 0.05, control vs. SCF+G-CSF+ Mut NFκB. ♦♦ p < 0.01, ♦ p < 0.05, SCF+G-CSF+ Wt NFκB vs. SCF+G-CSF+ Mut NFκB. Mean ± SE, n=3.
Figure 5.
SCF+G-CSF-induced neurite outgrowth is regulated by NFkB.
(A-D) Neurite outgrowth was examined 24h after seeding. (E-H) Neurite outgrowth was determined 72h after seeding. Cortical neurons were cultured for 2h, and NFkB inhibitor, Bay 11-7082 (Bay) (10µM for A-D, 5µM for E-H), was then added 5min (A-D) or 60min (E-H) before SCF+G-CSF treatment. (A-C, E-G) Representative confocal images of the neurites on the underside of transwell membranes. (D and H) Bar graphs represent quantification of neurite outgrowth with different treatment. Note that the SCF+G-CSF-induced neurite extension is significantly prevented by the NFkB inhibitor. Scale bars, 50µm. ** p < 0.01, Mean ± SE, n=4.
Figure 6.
Live neuron images for neurite outgrowth.
Note that SCF+G-CSF treatment leads to a marked increase in neurite extension, and that NFκB inhibitor, BAY 11-7082, prevents the SCF+G-CSF-induced the enhancement of neurite outgrowth. The images were selected at different time points from a movie recording neurite outgrowth during 8-26h after seeding (see Movies S1, S2, and S3).
Figure 7.
SCF+G-CSF upregulates BDNF and NGF at both the levels of transcription (mRNA) and translation (protein).
(A and B) BDNF and NGF gene expression after SCF, G-CSF, or SCF+G-CSF treatment. Quantification of BDNF and NGF gene expression was performed with Real-Time PCR. (A) Quantification data for BDNF gene expression. Note that SCF or SCF+G-CSF induces a significant increase in BDNF gene expression at both 6 and 24h after treatment as compared to medium controls (**p < 0.01). The levels of BDNF gene expression are much higher in SCF+G-CSF-treated neurons than those in SCF or G-CSF-treated at both 6 and 24h after treatment (◆◆p<0.01). SCF also causes a significantly higher level of BDNF gene expression than those of G-CSF at 6 h post-treatment (◆◆p<0.01). G-CSF only induces an increase in BDNF gene expression at 24h (**p < 0.01) but not at 6h post-treatment as compared to medium controls. SCF-, G-CSF- (24h only), or SCF+G-CSF-induced upregulation of BDNF gene expression is significantly blocked by BAY 11-7082 (●● p < 0.01). (B) Quantification data for NGF gene expression. Note that both SCF and SCF+G-CSF treatments result in a significant upregulation of NGF gene expression 6 and 24 h post-treatment when compared to medium controls (** P < 0.01) or G-CSF (◆◆p<0.01). BAY 11-7082 significantly prevents SCF- or SCF+G-CSF-induced upregulation of NGF gene expression (●● p < 0.01). Mean ± SD, n=3-4. (C and D) SCF+G-CSF-induced the upregulation of BDNF and NGF gene expression is prevented by siRNAs against BDNF (C) or NGF (D). Gene expression was examined 36h after adding siRNAs. Neg: negative controls for siRNA. **p < 0.01. Mean ± SE. N=3-4. (E-H) SCF+G-CSF-induced increases of BDNF and NGF protein production are inhibited by siRNAs against BDNF (E, G) or NGF (F,H) 72h after adding siRNAs to neurons. (E, F) Representative Western-Blot bands for BDNF (E) and NGF (F) proteins after different treatments. S: SCF. G: G-CSF. S+G: SCF+G-CSF. Neg: negative controls for siRNAs. (G and H) Semi-quantification of Western-blot data for BDNF (G) and NGF (H) proteins. *p < 0.05. Mean ± SE, n=3-4.
Figure 8.
The involvement of BDNF in SCF+G-CSF-induced enhancement of neurite outgrowth.
(A) The role of BDNF and NGF on SCF+G-CSF-induced enhancement of neurite outgrowth. Neurite outgrowth was quantified with a Neurite Outgrowth Quantification kit 72h after introducing siRNAs to neurons. SCF and G-CSF were added to neurons one hour after siRNA transfection. Note that SCF+G-CSF treatment results in a significant enhancement of neurite outgrowth. In addition, SCF+G-CSF-induced neurite outgrowth is significantly blocked by BDNF siRNA or BDNF siRNA+NGF siRNA, whereas BDNF siRNA+NGF siRNA treatment has no significant difference as compared to the BDNF siRNA treatment. NGF siRNA shows a trend towards decreasing SCF+G-CSF-induced neurite outgrowth. Negative siRNAs do not affect the SCF+G-CSF-induced the enhancement of neurite outgrowth as the neurite outgrowth in the negative siRNA-treated neurons still remains significantly greater than those of medium controls and BDNF siRNA+NGF siRNA. Neg: negative controls for siRNA. **p < 0.01, *p < 0.05. Mean ± SE, n=8. (B) SCF+G-CSF-induced enhancement of neurite outgrowth is mediated by BDNF receptor, TrkB. The neurite outgrowth was tested 72 h after treatment. Note that SCF+G-CSF-induced the increase in neurite extension (SCF+G-CSF vs. control, *p < 0.05) is significantly blocked by an anti-TrkB antibody (SCF+G-CSF vs. SCF+G-CSF+TrkB antibody, **p < 0.01). Scale bars, 50µm. Mean ± SE, n=3.