An investigation of gene expression in single cells derived from Nestin-expressing cells in the adult mouse midbrain in vivo

Abstract

Generation of new dopamine (DA) neurons in the adult midbrain is a controversial issue in development of better treatments for Parkinson's disease (PD). Previous research suggests Nestin-expressing neural precursor cells (NPCs) have a propensity to differentiate into neurons here, including DA neurons. In the present study we sought confirmation of this by studying gene expression in single Nestin-expressing cells and their progeny/ontogeny within the adult mouse midbrain. Cells were identified by administering a pulse of Tamoxifen to adult Nestin-CreERT2×R26eYFP transgenic mice. Samples of cytoplasm were harvested 4 days to 8 months later from individual eYFP+ cells in acutely prepared midbrain slices and analysed by RT-qPCR for gene expression. Remarkably, most eYFP+ cells co-expressed genes associated with mature (including DA) neurons (i.e. NeuN, Gad1, Gad2, vGlut2, TH and/or D2R) and neurogenesis (i.e. Ki67, Dcx, Ncam, Pax6, Ngn2 and/or Msx1), and this was true at all time-points following Tamoxifen. Indeed, cell proliferation genes (Nestin, Ki67) were exclusively expressed by eYFP+ cells with mature neuronal morphology and gene expression, and only at early time-points after Tamoxifen. Expression of proneuronal genes (Pax6, Msx1, Ngn2) was, however, higher in eYFP+ cells with immature morphology compared with mature morphology. Gene expression bore no relationship to cell location indicating that, in contrast to development, Nestin-expressing cells arise throughout the midbrain parenchyma and do not migrate long distances. On the other hand, gene expression did change with time after Tamoxifen, although not in a way consistent with neurogenesis. Overall, our results suggest that Nestin expression in the adult midbrain occurs in mature neurons, casting doubt on the premise of neurogenesis from Nestin+ NPCs here.