Foxa2 and Nurr1 Synergistically Yield A9 Nigral Dopamine Neurons Exhibiting Improved Differentiation, Function, and Cell Survival

Abstract

Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/ stem cell-based therapy of Parkinson's disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non-dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain-specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)-type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain-type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell-based therapy for PD. STEM CELLS 2010;28:501-512

Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage

“…Here, we showed that co-expression of the anti-apoptotic factor Bcl-xL along with BAM (BAMX) led to iNPCs that were highly proliferative over a long period of time and grew over 100 passages without alterations in major NPC properties. Numerous roles of Bcl-xL in NPC survival and maintenance have been documented previously in knock-out mouse embryonic brains (16), in vitro with gain-of-function studies using NPC cultures (30,31), and in clonal analyses using mouse embryonic neural precursor cells transduced with Bcl-xL (data not shown). To investigate whether the observed effects of Bcl-xL were due to its pro-survival functions, we analyzed the effect of Bcl-xL on the conversion of fibroblasts to iNPCs using dimerization-and mitochondrial localization-deficient Bcl-xL mutants (18).…”

In vitro generation of mature dopamine neurons by decreasing and delaying the expression of exogenous Nurr1

“…In addition, exogenous Nurr1 failed to induce markers specific to the midbrain (Lee et al, 2010). These issues could be addressed by co-expressing Foxa2, known to induce the expression of numerous midbrain-specific genes and facilitate Nurr1-induced DA neuron differentiation (Lee et al, 2010). As Foxa2 has a different expression pattern from Nurr1 during midbrain development, this strategy requires the development of another efficient inducible system, in which the expression of multiple genes could be controlled simultaneously but independently, based on their individual developmental patterns.…”

“…S9). In addition, exogenous Nurr1 failed to induce markers specific to the midbrain (Lee et al, 2010). These issues could be addressed by co-expressing Foxa2, known to induce the expression of numerous midbrain-specific genes and facilitate Nurr1-induced DA neuron differentiation (Lee et al, 2010).…”

Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation

“…3C). Because total cell numbers were not significantly altered by shFoxa2 treatment (data not shown), the Foxa2 effect on cell proliferation (Lee et al, 2010) is not likely to have influenced the DA gene expression data, although this possibility cannot be completely excluded. Forced Nurr1 expression yielded a few TH + DA cells in NPC cultures derived from non-dopaminergic embryonic cortical tissues (Fig.…”