An anatomical and single-cell gene expression characterisation of putative neurogenesis from nestin-expressing cells in the adult mouse midbrain
AffiliationFlorey Department of Neuroscience and Mental Health
Centre for Neuroscience
Document TypePhD thesis
Access StatusThis item is currently not available from this repository
© 2016 Dr. Parisa Farzanehfar
Due to significant midbrain dopamine (DA) cell loss in Parkinson’s disease (PD), it is generally believed the most effective and long-term treatment for PD motor symptoms will be midbrain DA cell replacement therapy, either by endogenous repair or cell transplantation. However, cell transplantation is hindered by failure of acquisition and maintenance of the DA phenotype by cells transplanted into the adult midbrain, and endogenous repair has not advanced very far because there appears to be very little or no neurogenesis and DA neurogenesis here. Evidence suggests Nestin-expressing neural precursor cells (NPCs) may give rise to neurones, including DA neurones in the adult midbrain, however this needs to be confirmed and underlying mechanisms established. The aims of my study were to: (1) Determine whether there is baseline neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain. (2) Investigate the gene expression profile of Nestin+ cells during different stages of putative ontogenesis in the adult mouse midbrain. (3) Test the possibility that putative neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain can be regulated by exogenous factors. To achieve these aims Nestin-expressing cells in the adult mouse midbrain were permanently labelled with enhanced yellow fluorescent protein (eYFP) by administering Tamoxifen to adult transgenic Nestin-CreERT2 x R26eYFP mice. Four-days to 8-months later eYFP+ cells were studied using a combination of whole-cell electrophysiology, single-cell qPCR and immunohistochemistry. eYFP+ cells expressed a range of genes consistent with birth, migration, neuronal and DA neuronal differentiation. Parsing the gene expression profiles of eYFP+ cells by their location in the midbrain indicated they arise anywhere throughout the midbrain and differentiate and integrate locally, rather than migrate long distances to populate midbrain nuclei. Most eYFP+ cells expressed mature neuronal genes, which was consistent also with their neuronal electrophysiology comprising action potentials and spontaneous post-synaptic currents. In comparison to neighbouring control (eYFP-) cells, eYFP+ cells expressed more immature neuronal genes (Pax6, Ngn2 & Msx1), which was also consistent with their more immature electrophysiology comprising hyper-excitability, hyperpolarized resting membrane potential, shorter duration sPSCs, and smaller membrane capacitance. Many (53%) eYFP+ cells expressed a combination of mature and immature neuronal genes at all time-points following Tamoxifen (an approximation of their age), including very ‘young’ cells (<2 months). Many (42%) of these young eYFP+ cells also had mature neuronal morphology (large cells with prominent processes) and electrophysiology. Nestin and Ki67 (a marker of cell division) were only expressed by large cells, and early on after Tamoxifen. Additionally, eYFP+ cells increased in number and size with time following Tamoxifen, and they decreased in number following administration of the anti-mitotic drug, Cytarabine (Ara-C). These data indicate that eYFP+ cells are capable of cell division. The level of expression of mature neuronal genes in eYFP+ cells also increased over a time-course of 7 months. In these respects, eYFP+ cells appear to proliferate, grow and differentiate into neurones, albeit slowly. To achieve the third aim of the study, bioinformatics analyses of the genes I identified as uniquely characterising eYFP+ cells (i.e. the proneuronal genes Pax6, Msx1 and Ngn2) highlighted valproic acid (VPA) as a drug that might regulate putative neurogenesis and DA neurogenesis from Nestin+ cells in the adult mouse midbrain. Infusion of VPA directly into the midbrain of adult mice for 2 weeks had no effect on the number of eYFP+ cells but significantly reduced the number of Pax6+, Pax6+/NeuN+ and eYFP+/NeuN+ cells. However, it also significantly reduced the number of NeuN+ cells generally (eYFP+ and eYFP-), pointing to a general effect of VPA on NeuN expression rather than neurogenesis from Nestin+ cells. Thus neither VPA nor targeting Pax6 appears to regulate of the number of eYFP+ cells or their differentiation into neurones in the adult mouse midbrain. In summary, my findings add to evidence of a small but significant population of Nestin-expressing cells in the adult mouse midbrain. While the origin of these cells remains unclear, my data suggest that: (1) some eYFP+ cells in the adult midbrain do undergo neurogenesis, albeit slowly; (2) Nestin expression in the adult midbrain is not limited to NPCs and classical neurogenesis, but also occurs in mature neurons, perhaps in relation to dendritic remodelling, cell death, or some other form of neural plasticity. 3) Neither VPA nor targeting Pax6 appears to regulate of the number of eYFP+ cells or their differentiation into neurones in the adult mouse midbrain. Further study of these cells may provide crucial information to assist DA cell replacement therapy for PD.
KeywordsParkinson's Disease; midbrain; Substantia Nigra (SN); single-cell PCR; Nestin-expressing cells
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