Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors

Abstract

Background: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA) and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR) by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains.

Methodology/principal findings: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice defective of NMDA receptor subunit-1, nicotine was still effective in significantly inhibiting the proliferation.

Conclusions/significance: Functional α4β2 nAChR subtype would be constitutively expressed to play a role in the mechanism underlying the determination of proliferation and subsequent differentiation fate into a neuronal lineage in association with preferential promotion of Math1 expression in undifferentiated neural progenitors of developing rodent neocortex independently of NMDA receptor activation.

Figure 1. Validation of procedures for neural progenitor isolation.

Neocortex was dissected from fetal rat brains, followed by preparation of lower cell layer fractions and subsequent culture with EGF for 12 days under floating conditions. Cells were then dispersed and cultured without EGF for 6 days under adherent conditions. Cells were fixed for immunocytochemistry analysis on the progenitor marker nestin, the neuronal marker lMAP2 and astroglial marker GFAP.

Figure 2. Expression of nAChR subunits in undifferentiated rat neural progenitors.

Neocortex was dissected from fetal rat brains, followed by preparation of the lower cell layer fractions. (A) Total RNA was extracted from cells before or after the culture in the presence of EGF for 12 consecutive days for RT-PCR analysis. Adult rat whole brain was used as a positive control. Typical pictures are shown with similar results in three independent sets of experiments. Undifferentiated progenitors were cultured with EGF in the presence of nicotine at different concentrations for determination of (B) the total size during culture, (C) MTT reduction, (D) LDH release and (E) the ratio of PI-positive cells cultured for 12 days. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine.

Figure 3. Effects of mecamylamine on cellular viability in rat progenitors.

Undifferentiated progenitors were cultured with EGF in either the presence or absence of 10 µM nicotine and 10 µM mecamylamine for a period up to 12 days for determination of (A) neurosphere size, (B) MTT reduction, (C) LDH release and (BD) the ratio of PI-positive cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^#P<0.05, significantly different from the value obtained in cells exposed to nicotine alone.

Figure 4. Differentiation of rat progenitors.

Cells were dispersed after the culture with EGF in either the presence or absence of 10 µM nicotine for 12 days, followed by further culture for an additional 6 days. Cells were then fixed for double immunocytochemical detection of both MAP2 and GFAP together with Hoechst33342 staining.

Figure 5. Effects of nicotine on differentiation of rat progenitors.

Cells were dispersed after the culture with EGF in either the presence or absence of 10 µM nicotine for 12 days, followed by further culture in (A) the absence and (B) presence of differentiation inducers such as ATRA and CNTF for an additional 6 days. Cells were then fixed for double immunocytochemical detection of both MAP2 and GFAP, followed by counting of the number of individual immunoreactive cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the value obtained in cells exposed to nicotine alone.

Figure 6. Expression of nAChR subunits in undifferentiated mouse neural progenitors.

Neocortex was dissected from fetal mouse brains, followed by preparation of the lower cell layer fractions. (A) Total RNA was extracted from cells before or after the culture with EGF for 10 consecutive days. Adult mouse whole brain was used as a positive control. Typical pictures are shown with similar results in three independent sets of experiments. Undifferentiated progenitors were cultured with EGF in the presence of nicotine at different concentrations for 10 days for determination of (B) the total size during culture and (C) the ratio of PI-positive cells cultured for 10 days in neurospheres. **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. (D) Undifferentiated progenitors cultured with EGF for 10 days were loaded with fluo-3, followed by cumulative addition of nicotine at a concentration range of 10 µM to 1 mM in either the presence or absence of DHβE at 10 µM and 1 mM and subsequent determination of the fluorescent intensity every 2 min. The individual fluorescence was normalized with the maximal fluorescence intensity obtained after addition of the Ca²⁺ ionophore A23187 at 10 µM.

Figure 7. Effects of nAChR antagonists in undifferentiated mouse neural progenitors.

(A) The selective nAChR antagonists DHβE, TC2559 and MLA were included in culture medium for determination of the total size of neurospheres during culture. (B) Cells were cultured with EGF in either the presence or absence of 10 µM nicotine and 10 µM DHβE for 10 days, followed by extraction of total RNA and subsequent RT-PCR analysis on cyclinD1 gene. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^#P<0.05, ^##P<0.01, significantly different from the value obtained in cells exposed to nicotine alone.

Figure 8. Expression and phosphorylation of particular proteins in undifferentiated mouse progenitors exposed to nicotine.

Undifferentiated mouse progenitors were cultured with EGF for 10 days and subjected to (A) Western blotting and (B) immunocytochemistry for α4 and β2 subunits of nAChRs. Immunocytochemical images are also shown with cells not treated with the primary antibody. (C) Undifferentiated cells were also exposed to 10 µM nicotine for 5 to 10 min for subsequent Western blotting. Typical pictures are shown with similar results in three independent sets of experiments.

Figure 9. Differentiation of mouse progenitors.

Cells were dispersed after the culture with EGF in either the presence or absence of 10 µM nicotine for 10 days, followed by further culture for an additional 4 days. Cells were then fixed for double immunocytochemical detection of both MAP2 and GFAP together with Hoechst33342 staining.

Figure 10. Effects of nicotine on differentiation of mouse progenitors.

(A) Cells were cultured with EGF in either the presence or absence of 10 µM nicotine, 10 µM DHβE, 10 µM TC2559 and 10 µM MLA for 10 days, followed by dispersion after removal of EGF and subsequent double immunocytochemistry analysis along with Hechst33342 staining for counting the number of immunoreactive cells. (B) Cells were cultured with EGF for 10 days in either the presence or absence of 10 µM nicotine, followed by extraction of total RNA and subsequent RT-PCR analysis on different bHLH genes. (C) Cells were cultured with EGF in either the presence or absence of 10 µM nicotine and 10 µM DHβE, followed by extraction of total RNA and subsequent RT-PCR analysis on Math1 gene. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the control value obtained in cells exposed to nicotine alone.

Figure 11. Neural progenitors prepared from NMDAR1-null mice.

Neocortex was dissected from WT (NR1^+/+) and NMDAR1-null (NR1^−/−) mice, followed by culture with EGF for 10 days and subsequent determination of (A) the total size of neurospheres and (B) the number of individual immunoreactive cells on double immunocytochemistry analysis along with Hechst33342 staining. *P<0.05, **P<0.01, significantly different from the value obtained cells from WT mice. Cells from WT and NMDAR1-null mice were also cultured with EGF in either the presence or absence of 10 µM nicotine, 10 µM DHβE and 10 µM MLA for 10 days, followed by determination of (C) MTT reduction, (D) neurosphere size and (E) PI-positive dead cells. *P<0.05, **P<0.01, significantly different from each control value obtained in cells not exposed to nicotine. ^##P<0.01, significantly different from the control value obtained in cells exposed to nicotine alone.