Universität Tübingen
Establishment and Characterization of a Human in vitro Cell Model for Parkinson’s Disease
Abstract
A major obstacle on the way to understand the molecular pathogenesis of Parkinson’s disease (PD) and to develop disease-modifying treatments is the lack of suitable model systems that capture the relevant molecular events leading to human disease and are also accessible to compound screening. This cumulative thesis describes the establishment of two novel cellular model systems based on induced pluripotent stem cells (iPSCs): the generation of an in vitro cell model for PD consisting of post-mitotic midbrain dopaminergic neurons, and the derivation and expansion of human neural progenitors for neurodegenerative disease modelling. For the first model, fibroblasts from two female PD patients with a G2019S mutation in the LRRK2 gene and four age- and sex-matched controls were reprogrammed into iPSCs. This was achieved through infection with the four factors Oct-4, Sox-2, Klf-4 and c-myc published by Shinia Yamanaka and isogenic gene-controls were generated using zinc finger nucleases (ZFNs) that differed from the original iPSCs only in the DNA base responsible for the G2019S mutation. Finally, one gene-corrected wild-type-LRRK2 line was obtained for each G2019S-LRRK2 line. In addition, an isogenic G2019S-LRRK2 line was generated for the wild-type-LRRK2 control C4, also through ZFN technology. The iPSCs were then differentiated into midbrain dopaminergic (mDA) neurons by an optimized differentiation protocol. Several studies on these mDA neurons showed that they were electrophysiologically active and expressed neuronal as well as dopaminergic neuronal markers. To clarify whether phenotypic changes can be observed, mDA neuronal cultures were investigated for two already described phenotypes associated with LRRK2: reduced neurite outgrowth and increased sensitivity to oxidative stress. Indeed, a reduced velocity of outgrowing neurons could be observed in G2019S-mutated iPSC-derived neurons. In addition, an increased sensitivity of the mutant neurons against oxidative stress could also be confirmed. As such, these assays suggested that iPSC-derived neurons from PD patients can serve as an in vitro PD model. For further characterization of this PD model, the expression of the proteins alpha-synuclein and TAU or pTAU was investigated, as these proteins are accumulated or hyperphosphorylated in the brain of PD patients. An accumulation on protein level could be stated for both proteins as well as an elevated RNA level for TAU. Mechanistic causes of these phenotypes such as the specific dysregulation of certain genes and an increased activation of Erk1/2 by G2019S-LRRK2 are described in the shared publication of Peter Reinhardt and me from 2013 in Cell Stem Cell: „Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression.“ The second cell model was generated to address the question of the suitability of iPSCs for high-throughput screenings. Due to relatively high heterogeneity, iPSC-derived neurons, as described above, are not ideally suited for this purpose. An additional aim was to develop a cell model that is not solely limited to PD but also allows studying other neurodegenerative diseases. Therefore, an iPSC-derived cell model system was established consisting of a robust neural progenitor cell type that is able to give rise to several neuronal subtypes. Results of this project were published in PLoS One in 2013: “Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling.”
Author and committee
dc:creator, dc:contributor.*- Author
-
- Schmid, Benjamin
Identifiers
dc:identifier.*- Identifier
- hdl:10900/49974