Evidence of functional duplicity of Nestin expression in the adult mouse midbrain

Abstract

Whether or not neurogenesis occurs in the adult substantia nigra pars compacta (SNc) is an important question relevant for developing better treatments for the motor symptoms of Parkinson's disease (PD). Although controversial, it is generally believed that dividing cells here remain undifferentiated or differentiate into glia, not neurons. However, there is a suggestion that Nestin-expressing neural precursor cells (NPCs) in the adult SNc have a propensity to differentiate into neurons, which we sought to confirm in the present study. Adult (>8-weeks old) transgenic NesCreER/GtROSA or NesCreER/R26eYFP mice were used to permanently label Nestin-expressing cells and their progeny with β-galactosidase (β-gal) or enhanced yellow fluorescent protein (eYFP), respectively. Most β-gal+ or eYFP+ cells were found in the ependymal lining of the midbrain aqueduct (Aq) and in the midline ventral to Aq. Smaller but significant numbers were in the periaqueductal gray (PAG), the ventral tegmental area (VTA), and in SNc. Low-level basal proliferation was evidenced by a modest increase in number of β-gal+ or eYFP+ cells over time, fewer β-gal+ or eYFP+ cells when mice were administered the anti-mitotic agent Cytarabine, and incorporation of the proliferation marker bromodeoxyuridine (BrdU) in a very small number of β-gal+ cells. No evidence of migration was found, including no immunoreactivity against the migration markers doublecortin (DCX) or polysialic acid neural cell adhesion molecule (PSA-NCAM), and no dispersal of β-gal+ or eYFP+ cells through the midbrain parenchyma over time. However, β-gal+ or eYFP+ cells did increase in size and express higher levels of mature neuronal genes over time, indicating growth and neuronal differentiation. In mice whose SNc dopamine neurons had been depleted with 6-hydroxy-dopamine, a model of PD, there were ~2-fold more β-gal+ cells in SNc specifically, although the proportion that were also NeuN+ was not affected. Remarkably, as early as 4days following putative Nestin-expression, many β-gal+ or eYFP+ cells had mature neuronal morphology and were NeuN+. Furthermore, mature neuronal β-gal+ cells were immunoreactive against the self-renewal or pluripotency marker sex determining region Y-box 2 (Sox2). Overall, our data support the notion that some Nestin-expressing, presumably NPCs, have a limited capacity for proliferation, no capacity for migration, and a propensity to generate new neurons within the microenvironment of the adult midbrain. However, our data also suggest that significant numbers of extant midbrain neurons express Nestin and other classical neurogenesis markers in contexts that are presumably not neurogenic. These findings foreshadow duplicitous roles for Nestin and other molecules that are traditionally associated with neurogenesis in the adult midbrain, which should be considered in future PD research.

Nestin-expressing progenitor cells: function, identity and therapeutic implications

“…Further, time-course analyses did not reveal classical neurogenesis evidenced by place of birth, neuronal differentiation, maturation and integration, raising the possibility that some mature neurons may express Nestin in midbrain [ 99 ]. Others have found hints of differentiation by means of cell growth and higher levels of expression of mature neuronal genes over long periods, albeit low proliferative rate and no evidence of migration [ 100 ]. Interestingly, single-cell gene expression data suggested that up-regulation of mature neuronal genes in Nestin - cre ERT2 -traced midbrain cells occurs over several months, not days, providing robust evidence for presence of Nestin + neural progenitors in adult midbrain.…”

“…Remarkably, 4 days after tamoxifen many Nestin - cre ERT2 -traced cells have mature neuronal morphology, and are positive for both neuronal nuclear antigen and the pluripotency marker Sox-2. This short timeframe may be not enough for Nestin + progenitors to become mature neurons, again raising the possibility that mature neurons express Nestin and other immature genes under certain conditions and in contexts that may be not neurogenic [ 100 ]. Other authors have selectively deleted tyrosine hydroxylase in Nestin- or Sox-2-traced cells using cre ERT2 systems.…”

Developmental pathway genes and neural plasticity underlying emotional learning and stress-related disorders

“…Although neurogenesis is a complex process regulated by a vast array of molecules, the functional heterogeneity of many of these regulators make them ill-suited to attempting to modulate neurogenesis without altering other forms of plasticity in multiple brain regions. For instance, neurotrophins such as brainderived neurotrophic factor, permissive plasticity factors such as polysialylated neural cell adhesion molecule, and even other stem-cell markers like nestin, while crucial to neurogenic function, also appear to regulate diverse forms of plasticity in other brain areas (Guirado et al 2014;Ehrlich and Josselyn 2016;Farzanehfar et al 2017). Conversely, the highly regionalized nature of TLX expression in the adult brain makes this transcription factor an attractive candidate for future studies on its role in mediating neurogenesis directly and specifically, particularly in light of recent evidence suggesting that TLX may be a point of crosstalk between the neuroinflammatory response and reductions in hippocampal neurogenesis observed following stress (Ryan et al 2013).…”

Nestin in immature embryonic neurons regulates axon growth cone morphology and Semaphorin3a sensitivity

“…Nestin mRNA in neurons would be an indicator that nestin is actually being translated in early neurons. Several paper have described nestin mRNA in neurons in developmental, normal adult, and pathological/injury contexts (Crino & Eberwine 1996;Zhu et al 2011;Kuo et al 2005;Arner et al 2015;Perry et al 2012;Farzanehfar, Lu, et al 2017;Dey et al 2017;Farzanehfar, Horne, et al 2017;Bigler et al 2017), thus nestin expression is not excluded from neurons. Nestin's presence in subpopulations of adult neurons suggests that some neurons may never lose nestin expression, or may regain it later.…”