Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells

Abstract

Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult.

In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multi-passaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Over-expression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers.

Introduction

Mesencephalic dopamine (mesDA) neurons are generated in the mouse ventral midbrain (VM) during a restricted time period starting at embryonic day E10 [1]. Typically, dopamine neurons are detected by the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the dopamine (DA) pathway. In the midbrain, the dopaminergic neurons and their progenitors also express genes such as Nurr1, Lmx1a, Lmx1b, Pitx3, En1, En2, and Ngn2, which are necessary for correct specification and/or survival in vivo (reviewed in [2], [3]) and in vitro [4], [5], [6].

Previous studies have demonstrated that over-expressing Nurr1, an orphan nuclear receptor that is essential for DA differentiation, may regulate TH expression in cultured cells [6], including embryonic stem (ES) cells, primary neural precursors derived from cortex, midbrain and developing striatum [7], [8], [9], and in non-neural cells [10]. However, this potential has not yet been demonstrated in multi-passaged VM progenitor cultures. Additionally, the Nurr1-induced TH-expressing neurons from fetal cells are often phenotypically and functionally immature [9] and expression of other genes and proteins that are characteristic of a midbrain identity has not been firmly established in these cells.

Transcription factors belonging to the basic helix–loop–helix (bHLH) gene family promote neuronal differentiation (for review see [11]). In addition to acting as neuronal determinants, the proneural bHLH genes play a role in cell fate specification in several areas of the developing CNS [12]. Several members of the proneural gene family have recently been reported to be expressed in dividing mesDA progenitors residing in the developing ventral midbrain [13], [14] but only Ngn2 is required for the correct development of the mesDA neurons [13], [15]. While the potential for Ngn2 to promote neuronal fates from neural progenitors in vitro is well established [16], its ability to promote a mesDA fate from expanded VM progenitors has not been elucidated.

In this study, we analyzed the ability of Ngn2 and Nurr1 to induce formation of mesDA neurons from multi-passaged neurosphere cultures derived from fetal VM. Neural stem and progenitor cells are expanded in these cultures. However, while they are isolated from the VM at a developmental time-point when mesDA neurons are actively generated, the neurons generated upon differentiation from these expanded VM progenitors never express TH or other markers characteristic of a dopaminergic phenotype (own observation and also reported in [17], [18], [19], [20]).

We show that over-expression of either Ngn2, Nurr1 or both combined, have distinct effects on neuronal differentiation in VM progenitor cell cultures. When over-expressing Ngn2 alone in multi-passaged VM-derived neurospheres we observed a significant increase in neuronal differentiation, however, the neurons did not express TH. On the other hand, over-expression of Nurr1 alone produced TH-positive cells with relatively immature neuronal morphologies that did not express any additional mesDA markers. In contrast, when Ngn2 and Nurr1 were over-expressed in the same culture, morphologically mature TH-positive neurons that also expressed additional dopaminergic and mesencephalic neuronal markers were generated. These data suggest that expanded VM progenitors that have lost their potential to generate mesDA neurons can be promoted to generate these cells by viral gene delivery in vitro.