Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits

Abstract

Background: Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown.

Methodology/principal findings: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro.

Conclusions/significance: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Figure 1. Glycine-induced Ca²⁺ signalling in fura-2 loaded NPCs differentiated for 1 or 3 weeks.

A, Changes in [Ca²⁺]_i were evoked by application of 100 µM glycine (Gly), 100 µM D-serine (D-Ser), 20 µM strychnine (Stry), and 50 mM KCl in cells differentiated for 3 weeks. The trace is the mean response of eight cells from a representative experiment. B, Summary of the [Ca²⁺]_i response amplitudes (n = 22–27 cells; means ± SEM) and fractions of cells (n = 60–75 from 86 cells) responding to different stimuli was obtained from 4 experiments as shown in A. C, NPCs differentiated for 1 week showed smaller increases in [Ca²⁺]_i upon application of 100 µM glycine and 50 mM KCl. The trace is the mean response of 8 cells from a representative experiment. D, Fractions of cells differentiated for 1 week (n = 7–29 of 139 cells) or 3 weeks (n = 60–75 from 86 cells) responding to different stimuli were obtained from 5 and 4 experiments as shown in A and C, respectively.

Figure 2. Functional analysis of glycine receptors in human midbrain-derived NPCs differentiated for 3 weeks in vitro.

A, Whole-cell recordings of currents elicited by applications of increasing glycine concentrations (10 µM – 10 mM). B, Glycine dose-response curve (EC₅₀ = 99.2 µM, hillslope = 0.95, n = 10) indicates a glycine EC₇₀ of 300 µM that was used in further electrophysiological experiments.

Figure 3. Pharmacological characterisation of glycine receptors in NPCs differentiated for 3 weeks.

A, Whole-cell currents evoked by a glycine EC₇₀ were all markedly blocked by strychnine and partly inhibited by picrotoxin, pregnenolone sulphate and a high bicuculline concentration. In contrast to the positive modulation by 10 µM ZnCl₂, a reduction of glycine currents was induced by 100 µM ZnCl₂. Also, co-application of a glycine EC₇₀ and tropisetron showed a positive modulatory effect. This pharmacological profile suggests receptor isoforms containing α2β subunits (B, n = 9–10; all data are given as means ± S.E.M.).

Figure 4. Real time PCR analysis of glycine receptor subunits expressed by NPCs after 1 and 3 weeks of differentiation.

Quantitative real-time PCR (SYBR green assay) was performed for each transcript and control (β2-microglobulin). Cycle threshold (Ct) values were normalized to the Ct values of the control and are given as log2^−ΔCt (ΔCt = Ct – Ct β2-microglobulin). Data are presented as means ± S.E.M. of 3 independent experiments (3 NPC lines) each performed in duplicate. A, The bar graph shows predominant expression of α2 and β subunits in NPCs differentiated for 3 weeks that is significantly different from all other glycine receptor subunits; significant differences between log2^−ΔCt values are marked (***p<0.001, ANOVA and Newman-Keuls post test). B, After 1 week of cell maturation the various glycine receptor isoforms are not yet expressed to a significantly different extent. The expression of α2, α4 and β subunits was markedly lower in NPCs differentiated for 1 week than for 3 weeks.

Figure 5. Immunocytochemistry of human mesencephalic NPCs after 3 weeks of standard differentiation in vitro.

Photomicrographs of NPCs immunoreactive for MAP2 (A) and glycine receptor subunits (B); nuclei were counter-stained with DAPI (C). Merged picture illustrates that neuronal cells express glycine receptor subunits (D). Note, this glycine receptor antibody is specific for all subunits.