Molecular and cellular characterization of midbrain dopaminergic neuron development

Midbrain dopaminergic (mDA) development is a complex yet highly controlled mechanistic process that is conserved across species. The understanding of these molecular details can open windows to new avenues of therapeutic medicine. Parkinson’s disease (PD) is a debilitating neurological disorder that to date has no cure or established cause. With various aspects of mDA development being revealed, the aim for a permanent treatment of PD itself is getting closer. Included within this thesis are four papers and two manuscripts covering diverse points of mDA neuron development and PD.

In Paper I we explore the role of transcription factor Pbx1 to promote mDA differentiation through activation of Pitx3 and repression of Onecut2. Pbx1 is also involved in protection from oxidative stress through Nfe2l1, an important aspect of PD. In Paper II we explore the cellular diversity of the ventral midbrain through the use of single-cell RNA-sequencing. The cellular transcriptional profiles aid in revealing the mDA neuron lineage and a cross-species comparison of mouse and human. To conclude, we use molecular tools to evaluate stem-cell derived mDA preparations for cell replacement therapy (CRT) in PD. In Paper III we review the current knowledge of Wnt signaling related to mDA development and further investigate the human single-cell data set from Paper II for other possible Wnt components that have yet to be explored for their role in development. In Paper IV we explore the composition of the mDA cellular environment using RNA-sequencing data. Here we apply a novel approach to gain insight to specific contributions from various cell types to the extracellular matrix, its modulators, and signaling ligands. We find a transcription factor network centered around Arntl1 in radial glia type 1 cells, a putative progenitor to the neuronal lineage. In Paper V we investigate the matricellular protein R-spondin 2. As a Wnt signaling activator, we show R-spondin 2 has a role in mDA differentiation when applied to embryonic stem cell differentiation protocols. This has direct translational impact in CRT for PD. In Paper VI we explore the role of Wnt/planar cell polarity signaling in midbrain development. Specifically, we elucidate the roles of Ror2 and Vangl2 in mDA development and their participation in morphogenesis and neurogenesis.

In conclusion, this thesis encompasses research on midbrain development from molecular details at a single-cell level to cellular components affecting global developmental processes. Here I present findings to be included towards a greater understanding of midbrain development and novel ideas relevant to translational research in CRT for PD.

List of scientific papers

I. Villaescusa JC, Li B, Toledo EM, Rivetti di Val Cervo P, Yang S, Stott SR, Kaiser K, Islam S, Gyllborg D, Laguna-Goya R, Landreh M, Lönnerberg P, Falk A, Bergman T, Barker RA, Linnarsson S, Selleri L, Arenas E. A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease. The EMBO Journal. 2016 Sep 15;35(18):1963-78.
https://doi.org/10.15252/embj.201593725

II. La Manno G, Gyllborg D, Codeluppi S, Nishimura K, Salto C, Zeisel A, Borm LE, Stott SR, Toledo EM, Villaescusa JC, Lönnerberg P, Ryge J, Barker RA, Arenas E, Linnarsson S. Molecular Diversity of Midbrain Development in Mouse, Human and Stem Cells. Cell. 2016 Oct 6; 167(2):566-580.
https://doi.org/10.1016/j.cell.2016.09.027

III. Toledo EM, Gyllborg D, Arenas E. Translation of WNT developmental programs into stem cell replacement strategies for the treatment of Parkinson’s disease. British Journal of Pharmacology. 2017 May 26.
https://doi.org/10.1111/bph.13871

IV. Toledo EM, La Manno G, Rivetti di Val Cervo P, Gyllborg D, Islam S, Villaescusa JC, Linnarsson S, Arenas E. Molecular analysis of the midbrain dopaminergic niche during neurogenesis. bioRxiv. 2017 Jun 26. [Manuscript]
https://doi.org/10.1101/155846

V. Gyllborg D, Ahmed M, Toledo EM, Theofilopoulos S, Ffrench-Constant C, Arenas E. The matricellular protein R-Spondin 2 (RSPO2) promotes midbrain dopaminergic neuron neurogenesis and differentiation. [Manuscript]

VI. Gyllborg D, Salašová A, Toledo EM, Gao B, Yang Y, Villaescusa JC, van Amerongen R, Arenas E. Ror2 and Vangl2 control dopaminergic neurogenesis and multiple aspects of cell polarity in the midbrain floor plate. [Manuscript]