Junior researcher Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moskva, Russia
Abstract: The G2019S mutation in LRRK2 kinase is the most common genetic mutation among patients with Parkinson's disease (PD). Although the G2019S mutation is known to increase the kinase activity of LRRK2, more work is needed to determine which LRRK2-regulated biological processes are primarily responsible for the pathogenesis of PD. Using CRISPR/Cas9, we generated isogenic induced pluripotent stem cells (iPSCs) containing different combinations of LRRK2 alleles – G2019S/G2019S, G2019S/wt, wt/wt, knockout (KO)/wt, KO/KO. This set of iPSC lines represents the entire spectrum of LRRK2 kinase activity from maximal (G2019S/G2019S) to null (KO/KO) and can be a helpful tool to clarify abnormalities caused by increased LRRK2 activity. We differentiated these iPSCs into dopaminergic neurons (DAns), which are lost in PD progression, and performed omics analyses. Both transcriptomic and proteomic analyses detected upregulation of nucleic acid metabolic pathways in mutant DAns. Phosphorylation of some RNA-binding proteins correlated with LRRK2 kinase activity, suggesting their role as physiological substrates of LRRK2. Transcriptomic and proteomic data also revealed altered composition of extracellular matrix which was subsequently validated throughout the stages of neuronal differentiation. Furthermore, multi-omics results indicated upregulation of innate immune response correlated with LRRK2 kinase activity. In addition, we observed an aberrant store-operated calcium entry (SOCE) in DAns with the G2019S mutation. High SOCE is known to increase mitochondrial calcium level, so we hypothesized that immune response in mutant DAns might be caused by mitochondrial DNA (mtDNA) leakage. While exploring immune signaling in DAns, we showed that LRRK2 knockout DAns have higher basal expression of type-I interferon (IFN-I) and interferon-stimulated genes, whereas all isogenic DAns have impairments in IFN-I response after extracellular mtDNA treatment. We are now investigating the role of such impairments that may elucidate possible dysregulation of neuronal immune signalling in PD. Taken together, the diverse set of isogenic genome-edited iPSCs in combination with omics approaches helps identify early pathogenic consequences of the G2019S mutation associated with increased LRRK2 kinase activity.
Funding Source: This work was supported by grant 075-15-2019-1669 from the Ministry of Science and Higher Education of the Russian Federation.
