IL-6 mediates neural stem cell differentiation depending on its soluble receptor sIL-6R. (A) H-IL-6 enhances gp130 receptor expression in EGF-responsive neural stem cells in vitro. NSCs were grown in EGF (20 ng/ml), EGF (20 ng/ml) plus H-IL-6 (100 ng/ml), or EGF (20 ng/ml) plus IL-6 (100 ng/ml) for three passages (3P) as described in Materials and Methods. (B) Thereafter, lysates were prepared and Western blot was performed using the respective antibodies as indicated. NSCs were treated as in A. The presence of H-IL-6 (100 ng/ml) enhances differentiation of NSCs, thus mediating neurogliogenesis. H-IL-6 down-regulates the stem cell marker nestin while up-regulating the neural marker TUJ-1 as well as the glia markers GFAP (astrocytes) and MBP and KROX20 (oligodendrocytes). No change in caspase-3 or activated caspase-3 (due to apoptosis) expression could be detected; α-tubulin was used as control for equal sample loading and as reference protein for the quantitative analysis. (C) Quantitative analysis of the Western blots shown in A and B (*p < 0.05, compared with control). (D) Dose-dependent effect of H-IL-6 on NSCs. NSCs were grown as in A and tended to differentiate (decrease in nestin expression) if exposed to higher concentrations of H-IL-6 (0, 50, or 100 ng/ml H-IL-6). (E) Quantitative analysis of the Western blots shown in D (*p < 0.05, compared with control). (F) The classical neurotrophic factors NGF, BDNF, and NT-3 (100 ng/ml each) do not mediate NSCs differentiation in the presence of EGF as the neural stem cell marker nestin, and the differentiation markers TUJ-1 and GFAP are almost unchanged after culturing for 3P. (G) Quantitative analysis of the Western blots shown in F (*p < 0.05, compared with control).

IL-6 mediates neural stem cell differentiation by decreasing the nestin-positive population. (A) Immunocytochemical analysis of H-IL-6–treated NSCs. NSCs were first grown (3P) in EGF (20 ng/ml) before inducing differentiation in neurobasal medium (NB, plus B27 supplement) with H-IL-6 (100 ng/ml) or without (control) for 5 d. H-IL-6 enhances neurogliogenesis by up-regulating the neural marker TUJ-1 as well as the glia markers GFAP (astrocytes) and MBP (oligodendrocytes). GFAP staining indicates two morphological different types of glia cells (see also Figure 3A). Scale bar, 100 μm. (B) H-IL-6 differentiates nestin-positive NSCs. H-IL-6 mediates NSCs differentiation by reducing the number of nestin-positive cells. EGF (20 ng/ml) (control) and EGF plus H-IL-6 (100 ng/ml)-treated NSCs (grown for 3P) were made single by mechanical trituration. Immunocytochemistry was performed with a nestin antibody as described in Materials and Methods, and the nestin-positive cells were counted with a fluorescence microscope (Axiovert 200, Zeiss). Data are shown as mean ± SD of three independent experiments, each done in duplicate (**p < 0.01, compared with control). Scale bar, 20 μm. (C) Proliferation of NSCs is greatly reduced by H-IL-6. NSCs were first grown in EGF (20 ng/ml) for 3P before the proliferation assay (BrdU incorporation) was done. Quantification of BrdU incorporation is shown as mean ± SD of four independent determinations (**p < 0.01; H-IL-6,100 ng/ml plus EGF, compared with control, EGF only). (D) H-IL-6 inhibits NSCs proliferation in a dose-dependent manner as the neurospheres differentiate into neurons and glia cells. NSCs were grown as in C before the proliferation assay (ATP consumption) was done with various doses of IL-6 or H-IL-6 as described in Materials and Methods. Values represent the mean (±SD) from experiments done four times, each performed in triplicate (*p < 0.05, **p < 0.01; H-IL-6 compared with IL-6).

H-IL-6 mediates neurogliogenesis. (A) H-IL-6 mediates gliogenesis. NSCs were grown with EGF (20 ng/ml) in the presence of H-IL-6 (100 ng/ml) for 3P in vitro and then were differentiated for 5 d in NB (plus B27 supplement) media only (control) or in the presence of either H-IL-6 or IL-6 (each 100 ng/ml), and immunocytochemistry was performed using an anti-GFAP antibody as described in Materials and Methods. Differentiation of NSCs results in two morphological distinctive glia cell types. Scale-bar, 100 μm. (B) H-IL-6 mediates neurogenesis. NSCs were grown with EGF (20 ng/ml) in the presence of H-IL-6 (100 ng/ml) for 3P in vitro and then were differentiated as in A, and immunocytochemistry was performed using an anti-TUJ-1 antibody as described in Materials and Methods. H-IL-6–treated cells showed more neuronal branches than control or IL-6–stimulated cells. Scale-bar, 100 μm. (C) NSCs were grown only in EGF (control) or EGF plus H-IL-6 or plus IL-6 for 3P and were differentiated for 5 d, as indicated, in NB media (control group) or in the presence of H-IL-6 or IL-6. Western blot analysis shows the up-regulation of different neuronal subtypes upon differentiation with H-IL-6: TH (dopaminergic neurons), GLU-V (glutamatergic neurons), and GAD-65/67 (GABAergic neurons). (D) Quantitative analysis of the Western blots shown in C (*p < 0.05, compared with control). (E) NSCs were grown and differentiated as in C (control and H-IL-6 groups). Also with the immunocytochemistry method different neuronal subtypes could be detected if H-IL-6 was present during the differentiation process but were absent in control-differentiated cells. Scale-bar, 50 μm. (F) NSCs were grown and differentiated as in E. STMN2-specific immunocytochemistry (costaining with TUJ-1) revealed that H-IL-6–differentiated neurons display more branches than control neurons (for appropriate comparison, representative neurons were selected). Scale-bar, 50 μm. (G) NSCs were grown and differentiated as in E. Immunocytochemistry was done as in E with an anti-MAP2 antibody. MAP2-positive cells were counted in control and H-IL-6–treated cells. Data are shown as mean ± SD of four independent experiments, each done in duplicates (**p < 0.01, compared with control). (H) STMN2-positive cells from F were counted in control and H-IL-6–differentiated cells. Data are shown as mean ± SD of four independent experiments, each done in duplicates (**p < 0.01, compared with control).

Signaling pathway analysis of H-IL-6–activated NSCs neuronal-differentiation. (A and B) NSCs were grown in EGF (20 ng/ml) for 3P and then stimulated with H-IL-6 (100 ng/ml) or IL-6 (100 ng/ml) for 10 min as described in Materials and Methods before being subjected to Western blot. Results clearly show the activation of the JAK/STAT-3 and PKA/MAPK pathways. In addition, nuclear phosphorylated CREB (n) (CREB (c), cytoplasmic fraction) is significantly increased compared with control and IL-6–treated cells. cPKAα, catalytic subunit of protein kinase A α. (A) represents the quantitative analysis of the Western blots shown in B (*p < 0.05, compared with control). (C) Western blot is shown where NSCs were grown in the presence of the STAT-3 inhibitor cucurbitacin-I (Cucu-10/-100; 10 and 100 ng/ml) for 3P with EGF (20 ng/ml) and H-IL-6 (100 ng/ml). Almost all nestin- and GFAP-positive NSCs/progenitors seem to die (very low signal for either of them in the presence of Cucu-I), whereas TUJ-1–positive neuronal cells survive allowing neural precursors to differentiate upon H-IL-6 stimulation into neurons via activation/phosphorylation of MAPK/CREB (nuclear p-CREB (n)). (D) Quantitative analysis of the Western blots shown in C (*pp < 0.05, compared with control). (E) NSCs were grown in EGF for 3P and then stimulated with H-IL-6 as in A and B. The less specific STAT-3 inhibitor cucurbitacin-I (Cucu-10, 10 ng/ml) inhibits H-IL-6 (100 ng/ml)-activated CREB (phosphorylated fraction of nuclear CREB (p-CREB (n)) and STAT-3 phosphorylation. (F) Quantitative analysis of the Western blots shown in E (*p < 0.05, compared with control). (G) NSCs were grown in EGF and then stimulated with H-IL-6 as in A and B. Applying the specific STAT-3 signaling inhibitor (p-iP-STAT-3, 30 μM) still allows H-IL-6 (100 ng/ml) to stimulate the phosphorylation of STAT-3 but inhibits its action and thus GFAP activation. The presence of the inhibitor also permits CREB activation (phosphorylation, p-CREB) and by this neuronal (TUJ-1 up-regulation) differentiation and survival. (H) Quantitative analysis of the Western blots shown in G (*p < 0.05, compared with control; ^# p < 0.05, compared with H-IL-6–stimulated cells).

Ionic glutamate receptors in differentiated neural stem cells. (A) NSCs were grown only with EGF (20 ng/ml) for 3P in vitro and then differentiated in NB media plus B27 supplement for 5 d (without H-IL-6) as described in Materials and Methods. No ionic glutamate receptors were detected in these control-differentiated NSCs (n = 8). (B) NSCs were grown with EGF (20 ng/ml) in the presence of H-IL-6 (100 ng/ml) for 3P in vitro and then differentiated in the presence of H-IL-6 for 5 d as described in Materials and Methods. In H-IL-6–differentiated neural stem cells, glutamic acid-activated ionic receptors (non-NMDA receptors) were detected (n = 8). (C) H-IL-6–differentiated cells from B were further analyzed. The activation current was with AMPA characteristics as the selective noncompetitive AMPA antagonist GYKI-52466 could completely inhibit the AMPA-receptor-agonist–mediated effect. The membrane potential was held at −70 mV. The short dashed line indicates the baseline of zero current (n = 7). (D) Currents activated by NMDA in H-IL-6–differentiated cells from B were very small, 10 pA only. The short dashed line is the membrane potential. The bold lines indicate the addition of 200 μM NMDA plus 20 μM glycine or the addition of 200 μM AMPA. (E) The current voltage (I-V) relationships of NMDA and AMPA receptor channels of H-IL-6–differentiated cells from B.

IL-6—and depending on the presence of its soluble IL-6R—induces neural stem cells (NSCs) differentiation into astrocytes [via glia progenitor cells (GPC), pathway indicated by green arrows] and neurons [via neuronal progenitor cells (NPC), pathway indicated by red arrows]. During neurodegenerative processes apoptotic neurons release sIL-6R (purple receptor symbols) by shedding, whereas activated astrocytes release IL-6 (red circle). Via trans-signaling they can act on NSCs, which express (as GPCs and NPCs) very low or no IL-6 receptors but the common signaling receptor subunit gp130 (shown as green receptor-dimer symbols) that is used by all members of the IL-6-receptor family (Taga and Kishimoto, 1997; Heinrich et al., 2003). Although EGF and bFGF maintain NSCs' self-renewal, the IL-6–sIL-6R complex overrides those activities mediating NSCs differentiation. In contrast, classical neurotrophic factors such as NGF, BDNF, or NT-3 are unable to overcome EGF signaling. Increased gp130 expression on NSCs/NPCs and IL-6R-NGFR/TRKA (blue receptor symbols) transactivation by IL-6R/gp130 (white dotted line) enhances neuronal differentiation. Differentiated neurons express then various neurotrophic factors for their survival and proper functioning. Inhibiting the glia-differentiation pathway by, for instance, p-STAT-inhibitors (inhibition of nuclear transcription factor activity of p-STATs but not complete down-regulation of their expression) may turn H-IL-6 into an interesting potential therapeutic factor, acting on the MAPK/CREB signaling pathway, for the treatment of neurodegenerative diseases.