Astrocyte-derived interleukin-6 promotes specific neuronal differentiation of neural progenitor cells from adult hippocampus

Abstract

The purpose of this study was to investigate the ability of astrocyte-derived factors to influence neural progenitor cell differentiation. We previously demonstrated that rat adult hippocampal progenitor cells (AHPCs) immunoreactive for the neuronal marker class III beta-tubulin (TUJ1) were significantly increased in the presence of astrocyte-derived soluble factors under noncontact coculture conditions. Using whole-cell patch-clamp analysis, we observed that the cocultured AHPCs displayed two prominent voltage-gated conductances, tetraethyl ammonium (TEA)-sensitive outward currents and fast transient inward currents. The outward and inward current densities of the cocultured AHPCs were approximately 2.5-fold and 1.7-fold greater, respectively, than those of cells cultured alone. These results suggest that astrocyte-derived soluble factors induce neuronal commitment of AHPCs. To investigate further the activity of a candidate neurogenic factor on AHPC differentiation, we cultured AHPCs in the presence or absence of purified rat recombinant interleukin-6 (IL-6). We also confirmed that the astrocytes used in this study produced IL-6 by ELISA and RT-qPCR. When AHPCs were cultured with IL-6 for 6-7 days, the TUJ1-immunoreactive AHPCs and the average length of TUJ1-immunoreactive neurites were significantly increased compared with the cells cultured without IL-6. Moreover, IL-6 increased the inward current density to an extent comparable to that of coculture with astrocytes, with no significant differences in the outward current density, apparent resting potential, or cell capacitance. These results suggest that astrocyte-derived IL-6 may facilitate AHPC neuronal differentiation. Our findings have important implications for understanding injury-induced neurogenesis and developing cell-based therapeutic strategies using neural progenitors.

Figure 1

Differentiation of AHPCs under non-contact co-culture conditions (NCCC). AHPCs were cultured under four different culture conditions: (1) AHPCs cultured alone without astrocytes (AHPCs alone), (2) non-contact co-culture with astrocytes isolated from cerebral hemispheres of neonatal rat brain (NCCC with Brain-Astro), (3) non-contact co-culture with cortical astrocytes (NCCC with CTX-Astro) and (4) non-contact co-culture with hippocampal astrocytes (NCCC with HC-Astro). Differentiation of AHPCs was characterized using cell-type specific antibodies: TUJ1 for young neurons, RIP for oligodendrocytes and GFAP for astrocytes. Values represent the average percentage of AHPCs immunolabeled by each antibody. N (number of independent experiments) = 6 for AHPCs alone and N = 3 for each NCCC. Error bars represent standard error of the mean (SEM). Asterisks represent significant difference from the control culture of AHPCs alone (t test; *, p < 0.05; **, p < 0.0001). TUJ1 immunoreactivity of AHPCs under all NCCC conditions was significantly greater compared to AHPCs cultured alone. The average percentage of TUJ1-IR AHPCs co-cultured with hippocampal astrocytes was significantly greater than that of the AHPCs co-cultured with cortical astrocytes. The RIP-IR AHPCs were significantly increased only when the AHPCs were co-cultured with Brain-Astro, compared to the AHPCs cultured alone or co-cultured with either CTX-Astro or HC-Astro. The percentage of GFAP-IR AHPCs did not vary significantly under these conditions.

Figure 2

Comparison of electrophysiological properties of AHPCs cultured alone or co-cultured with neonatal astrocytes. AHPCs with neuronal-like morphologies were selected for whole cell recording in voltage-clamp mode. (A) AHPCs cultured in the absence of astrocytes under differentiation condition. (A1) A superimposed image of TUJ1-IR shown in red, GFP in green and DAPI-stained nuclei in blue. (A2) A representative whole cell recording trace showing sustained outward currents and small transient inward currents elicited by a family of depolarizing voltage steps (inset, magnified inward currents). (B) AHPCs co-cultured with astrocytes. (B1) A superimposed image of TUJ1-IR AHPCs. (B2) A representative trace displaying sustained outward currents and substantial transient inward currents. Scale bars in (A1) and (B1) represent 20 μm.

Figure 3

Interleukin (IL)-6 enhances expression of neuronal marker protein in AHPCs. (A) Average percentages (± SEM) of cell-type specific antibody labeling of AHPCs under three different NCCCs in the presence of goat anti-IL-6 neutralizing antibody (Anti-IL-6) or non-specific IgG produced from goat (goat IgG) as a control. Goat IgG was added at an equal concentration of Anti-IL-6 added (10 ng/ml). With addition of Anti-IL-6 to the hippocampal NCCC, TUJ1-IR AHPCs were significantly decreased (54.4% in control and 51.5% with goat IgG vs. 27.7% with Anti-IL-6). The blocking antibody had no effect on TUJ1 immunoreactivity in cortical NCCC (34.2% in control and 34.4% with goat IgG vs. 35.2% with Anti-IL-6). N (number of independent experimental sessions) = 3 for each co-culture session. Asterisks represent statistical difference at p < 0.05. NS, no significant difference. (B) Recombinant rat IL-6 (20 ng/ml) specifically induced neuronal differentiation of AHPCs (increased percentage of TUJ1 immunoreactivity). N = 6. The double asterisks represent a significant difference from the control culture (AHPCs alone) at p < 0.0001. The percentage of TUJ-IR AHPCs was significantly greater when cultured in the presence of purified IL-6, compared to that in the absence of IL-6 (38.5% with IL-6 vs. 13.1% without IL-6). Addition of Anti-IL-6 blocking antibody dramatically reduced the percentage of TUJ1-IR AHPCs when cultured with IL-6. (C) IL-6 enhanced primary neurite growth. AHPCs were cultured under differentiation condition for 6 days in the presence of various concentrations of purified IL-6 from 0 to 100 ng/ml (see ‘IL-6 treatment’ in ‘Methods and Materials’ section). The average length of primary neurites emanating from a TUJ1-IR AHPC was measured. From each condition of an experimental set, 17 to 23 TUJ1-IR AHPCs were analyzed. N = 3 independent experiments. Values are mean ± SEM. Asterisks indicate statistical difference (t test; *, p < 0.05; **, p < 0.0001).

Figure 4

IL-6 promotes the development of sodium currents in AHPCs undergoing differentiation. AHPCs were cultured alone without astrocytes under differentiation condition in the absence (A) or presence (B) of IL-6. AHPCs cultured in the presence of IL-6 developed membrane properties consistent with those of functional neurons. The AHPCs possessing neuronal morphologies were analyzed using whole cell patch clamping. (A) AHPCs cultured without IL-6. (A1) A superimposed image of TUJ1-IR AHPCs shown in red, GFP in green and DAPI-stained nuclei in blue. (A2) A representative family of traces showing TEA-sensitive sustained outward currents and very small transient inward currents elicited by depolarizing voltage steps (inset, magnified inward currents). (B) AHPCs cultured with IL-6. (B1) A superimposed image of TUJ1-IR AHPCs. (B2) A representative traces showing TEA-sensitive outward currents and larger TTX-sensitive, transient inward currents. (C) IV-plot showing the relationship between stimulating voltage and current density. (Upper) Recording showing TEA-sensitive outward currents (N = 5). (Lower) Recording showing TTX-sensitive inward currents (N = 3). Scale bars in (A1) and (B1) represent 20 μm.