Who is to blame? Investigating Genes within the 22q11.2 Deletion Region as potential Risk Factors for Parkinson's Disease using Human iPS Cells and CRISPR Perturbation
Final Report Abstract
Adults with genomic deletions on chromosome 22q11.2, which include a 3 megabase genomic region with approximately 41 protein-coding genes expressed in the human brain, have a higher risk of developing typical Parkinson's disease (PD); however, it is unclear which of these genes contribute to the neurodegenerative process. The goal is to identify genes in the 22q11 deletion that contribute to causal molecular mechanisms of neuronal dysfunction and predispose to a neurodegenerative process reminiscent of PD due to diminished gene dosage. (i) Using human induced pluripotent stem cells (iPSCs), I investigate whether iPSC-derived dopaminergic neurons from 22q11DS patients replicate pathophysiological phenotypes associated with PD. (ii) Implementing CRISPR perturbation will allow us to identify biological targets and pathways. I show successful differentiation of human iPSCs from 22q11DS patients and controls into neurons that express genetic markers for midbrain dopaminergic identity and neuronal maturation. In iPSC-derived neuronal progenitors, I found no differences in reactive oxygen species (ROS), ATP levels, or endolysosomal activity in cultures from 22q11DS compared to controls. By contrast, I detected increased levels of ROS in neurons (30 days in vitro) derived from 22q11DS patients compared to controls. I further detected enhanced endolysosomal activity with LysoTracker and increased ATP concentrations in 22q11DS neurons. Significant differences in mitochondrial function and density could only be detected between individual control and patient lines in neuronal progenitor cells and neurons (DIV30). Our findings are consistent with data from 22q11DS mouse models indicating increased ROS levels in cortical projection neurons and animal models of PD. The developed protocols for CRISPR perturbation in 22q11DS and control iPSCs will allow us to reversibly regulate gene expression and systematically increase protein levels of deleted genes in the 22q11.2 deletion in human iPSC-derived neurons to identify causative genes.
Publications
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A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell, 29(12), 1685-1702.e22.
Pantazis, Caroline B.; Yang, Andrian; Lara, Erika; McDonough, Justin A.; Blauwendraat, Cornelis; Peng, Lirong; Oguro, Hideyuki; Kanaujiya, Jitendra; Zou, Jizhong; Sebesta, David; Pratt, Gretchen; Cross, Erin; Blockwick, Jeffrey; Buxton, Philip; Kinner-Bibeau, Lauren; Medura, Constance; Tompkins, Christopher; Hughes, Stephen; Santiana, Marianita ... & Merkle, Florian T.
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Invesggagng genes within the 22q11.2 delegon region as potengal risk factors for Parkinson's disease in human iPSC models, Neuroscience, San Diego, United States (12-16 November 2022):Poster-Nr. 17A
Heinrich, L., Chen, M. Y. & Schüle, B.
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Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology. Journal of Neuroscience Methods, 378, 109653.
Heinrich, Laurin; Zafar, Faria; Morato, Torres C. Alejandra; Singh, Jasmine; Khan, Anum; Chen, Max Yang; Hempel, Cassandra; Nikulina, Nadya; Mulholland, Jonathan; Braubach, Oliver & Schüle, Birgitt
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Protocol for CODEX Fixagon Steps and Primary Angbody Staining for Induced Pluripotent Stem Cell-Derived Neurons v1.
Zafar, Faria & Morato Torres, C. Alejandra & Heinrich, Laurin & Singh, Jasmine & Hempel, Cassandra & Braubach, Oliver & Schuele, Birgie
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Automated 384-well SYBR Green Expression Array for Optimization of Human Induced Pluripotent Stem Cell Differentiation. BIO-PROTOCOL, 13(11).
Chen, Max; Heinrich, Laurin; Zafar, Faria; Sedov, Kamilla & Schuele, Birgitt
