Nuclear receptor and Wnt function in developing dopaminergic neurons

The progressive degeneration of dopaminergic neurons in the substantia nigra is one of the major pathological events associated with Parkinson’s Disease. Dopaminergic (DA) cell replacement therapy has emerged in recent years as a possible approach towards treating Parkinsonism. However, the knowledge of intrinsic and extrinsic regulators governing the development of DA progenitors for such strategies remains limited. This thesis examines the function of two major signaling players, nuclear receptors and Wnts and how they modulate DA development.

Nuclear receptors are important regulators of early developmental events and adult physiology. The orphan nuclear receptor Nurr1 has previously been shown to be essential for the acquisition of a dopaminergic neurotransmitter phenotype in the developing ventral midbrain. Herein, we provide genomic and physiological evidence that Nurr1 coordinates the expression of genes involved in survival and resistance to oxidative stress. We also found that the extracellular matrix protein, tenascin-C is strongly upregulated in Nurr1-expressing neural stem cells and that tenascin-C null mice exhibit an accelerated DA neurogenic program in vivo. These findings suggest that Nurr1 upregulates tenascin- C and maintains the Nurr1 precursor population in vivo. Combined, our data unravel a previously uncharacterized function of tenascin-C to delay the differentiation of Nurr1 precursors.

In another study, we investigated the function of two other nuclear receptors, liver X receptors (LXRs) α and β, in developing midbrain DA neurons. We demonstrate that loss of LXRα and LXRβ function results in diminished DA neurogenesis at the expense of an increased number of neuroepithelial cells and enhanced gliogenesis in vivo. Moreover, treatment of ventral mesencephalic, but not cortical progenitors with the LXR ligand 22-hydroxycholesterol reduced the number of RC2+ radial glia cells while simultaneously enhancing DA neurogenesis. These effects were completely abolished in LXRα/β-/- cells. These data show that LXRα and LXRβ coordinate neuronal versus glial cell-fate choices in ventral midbrain progenitors and illustrate a novel function for these nuclear receptors in DA neuron development.

The Wnt signalling pathway controls patterning, proliferation, migration, and cell differentiation events in the developing CNS. In this thesis we illustrate that ventral midbrain (VM) glia, but not cortical glia, secrete high levels of the lipoprotein Wnt5a. Additionally, VM glia express region-specific transcription factors such as Pax-2, En-1 and Otx-2 and increase the differentiation of cortical or VM Nurr1+ precursors into DA neurons. Blocking experiments using a Wnt5a blocking antibody indicate that the effects of ventral midbrain glia on Nurr1-positive neural precursors are partially mediated by Wnt5a. In summary, our results indicate that VM glia induce a DA phenotype in Nurr1- expressing neuronal precursors and that this effect is specific and involves the secretion of Wnt5a.

Recent expression analyses have indicated that additional Wnts, including Wnt2, may contribute to the development of DA neurons. However, the lack of available purified Wnt to address this has precluded such efforts. We describe the first successful purification and characterization of Wnt2. We find that purified Wnt2 promoted the differentiation of ventral precursor cultures into DA neurons. Treatment of DA cells with purified Wnt2 demonstrated that Wnt2 induced the accumulation of β-catenin and phosphorylation of Dishevelled-2. These signals could be inhibited by enzymatic removal of the post-translational lipid modification. Additionally, mice containing a targeted deletion of Wnt2 exhibit DA deficits at the onset of DA neurogenesis. These observations indicate that Wnt2 activates canonical Wnt signals and promotes the differentiation of DA precursors in vivo.

In summary, our data broaden our knowledge on the function of nuclear receptors and Wnts during DA development and raise the spectre of nuclear receptor and Wnt ligands that may be of clinical utility for stem cell-replacement strategies for PD.

List of scientific papers

I. Sousa KM, Mira H, Hall AC, Jansson-Sjostrand L, Kusakabe M, Arenas E. (2006). "Microarray analyses support a role for Nurr1 in resistance to oxidative stress and neuronal differentiation in neural stem cells." Stem Cells. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/17038671

II. Sousa KM, Hall AC, Steffensen K, Sacchetti P, Hazenberg C, Gustafsson JÅ, Arenas E. (1970). "Liver X Receptors regulate neuronal versus glial cell-fate choices in ventral midbrain dopaminergic progenitors." [Manuscript]

III. Castelo-Branco G, Sousa KM, Bryja V, Pinto L, Wagner J, Arenas E. (2006). "Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion." Mol Cell Neurosci 31(2): 251-62.
https://pubmed.ncbi.nlm.nih.gov/16243537

IV. Sousa KM, Ondr JK, Hofstra W, Castelo-Branco G, Bryja V, Bergman T, Lang RA, Arenas E. (1970). "Purified Wnt2 activates â-catenin and enhances the differentiation of dopaminergic precursors." [Manuscript]