Nicotinic acetylcholine receptors on NT2 precursor cells and hNT (NT2-N) neurons

This is the first report, to our knowledge, of prominent, natural expression of nAChR alpha4, alpha6 and alpha9 subunits in a human, neuronally-committed cell line. We performed studies with specific reference to the expression of nicotinic acetylcholine receptors (nAChR) to further characterize a human, postmitotic, transplantable, with a neuronal phenotype, cell line called hNT (also called NT2-N). hNT cells acquire a distinctive neuronal phenotype upon differentiation from their NT2 precursors. Immunocytochemical studies showed that NT2 cells were strongly immunopositive for alpha4 or alpha7 subunits, moderately immunopositive for alpha3/alpha5 subunits, and weakly immunopositive for beta2 or beta4 subunits, whereas hNT neurons showed positive, strong-to-moderate immunostaining for all of these nAChR subunits. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analyses indicated that levels of alpha7 subunit messages were similar in both NT2 and hNT cells, whereas alpha2, alpha10, and beta3 subunit transcripts were not detected. Levels of alpha3, alpha5, and beta4 subunit messages were lower in hNT neurons than in NT2 precursors. However, alpha4 and beta2 subunit messages were present in NT2 precursors but were greatly induced in hNT neurons. Levels of alpha6 and alpha9 subunit messages, not detectable in NT2 precursors, rose to high levels in hNT neurons. hNT cell nAChR subunit message levels were comparable to (alpha4, alpha5, beta4) or higher than (alpha6, alpha9, beta2) levels in adult human brain. NT2 and hNT cells may provide an excellent model for studies of neurogenesis, roles played by nAChR in differentiation and neurodegeneration, and effects of neuronal differentiation on nAChR expression.