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Published on: June 14, 2020
Basic Science and Pathogenesis
Jose Torrellas1, Aravindraja Chairmandurai1, Adamantios Mamais1
1University of Florida, Gainesville, FL, USA.
Background:
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, particularly the G2019S and R1441C variants, are the most prevalent genetic determinants of Parkinson's disease (PD). LRRK2 is increasingly recognized for its roles in immune signaling, with studies suggesting that interferon-gamma (IFNγ), a master immune regulator, may modulate its activity. Based on these findings, we hypothesize a synergistic interaction between LRRK2 mutations and IFNγ.
Method:
To explore this hypothesis, we utilized neurons derived from human induced pluripotent stem cells (iPSCs) harboring homozygous G2019S or R1441C LRRK2 mutations, with isogenic wildtype (WT) controls. After differentiation into neurons, cells were treated with recombinant human IFNγ or control media on day 17 of culture and harvested after 6 hours of treatment. Six experimental conditions were analyzed: (WT + IFNγ, WT + Control, G2019S + IFNγ, G2019S + Control, R1441C + IFNγ, R1441C + Control) We analyzed these experiments using immunoblotting and immunofluorescence methods to assess interferon-pathways, tau phosphorylation, and LRRK2 substrate engagement.
Result:
Using a dose-dependent paradigm, we optimized the minimum IFNγ dose to achieve maximal target engagement (STAT1 and phosphorylated-STAT1, pSTAT1). All three genotypes responded to IFNγ, producing equivalent levels of pSTAT1, confirming activation of the JAK-STAT pathway. We found that at baseline, levels of pSer202 tau (CP13) levels were moderately increased in both G2019S- and R1441C-LRRK2 relative to WT-LRRK2. IFNγ treatment increased CP13 levels in both G2019S- and R1441C-LRRK2, with the CP13 induction higher in G2019S-LRRK2 while WT-LRRK2 neurons did not show elevated CP13 following IFNγ treatment. IFNγ treatment phosphorylated Threonine73 on Rab10 (a LRRK2 substrate) more efficiently in G2019S-LRRK2 neurons compared to R1441C-LRRK2. We did not observe a genotype-dependent effect of IFNγ on phosphorylation of Ser106 on Rab12, another LRRK2 substrate.
Conclusion:
Our data indicates a LRRK2 genotype related response of LRRK2 neurons to IFNγ stimulus. This data lays the groundwork for future studies to dissect mechanisms of neuronal vulnerability in PD and the interplay between LRRK2 mutations and disease pathways.
Insights
Leucine-rich repeat kinase 2 (LRRK2) mutations in Parkinson's disease (PD) interact with interferon-gamma (IFNγ). LRRK2 G2019S and R1441C mutations show genotype-dependent responses to IFNγ, impacting tau phosphorylation and LRRK2 substrate engagement.
Area of Science:
- Neuroscience
- Genetics
- Immunology
Background:
- Leucine-rich repeat kinase 2 (LRRK2) mutations, specifically G2019S and R1441C, are major genetic factors in Parkinson's disease (PD).
- Interferon-gamma (IFNγ), a key immune regulator, is hypothesized to interact with LRRK2, potentially influencing PD pathogenesis.
Purpose of the Study:
- To investigate the synergistic interaction between LRRK2 mutations (G2019S, R1441C) and interferon-gamma (IFNγ) in human neurons.
- To explore the impact of IFNγ on LRRK2 activity, tau phosphorylation, and LRRK2 substrate engagement in different LRRK2 genotypes.
Main Methods:
- Utilized human induced pluripotent stem cell (iPSC)-derived neurons with homozygous G2019S or R1441C LRRK2 mutations and isogenic wildtype (WT) controls.
- Treated neurons with recombinant human IFNγ or control media and analyzed via immunoblotting and immunofluorescence.
- Assessed interferon-pathway activation (STAT1 phosphorylation), tau phosphorylation (CP13), and LRRK2 substrate phosphorylation (Rab10, Rab12).
Main Results:
- IFNγ activated the JAK-STAT pathway, indicated by equivalent STAT1 and pSTAT1 levels across all genotypes.
- Baseline tau phosphorylation (pSer202) was elevated in G2019S and R1441C neurons compared to WT.
- IFNγ treatment increased tau phosphorylation (CP13) in G2019S and R1441C neurons, with a more pronounced effect in G2019S.
- IFNγ enhanced Rab10 phosphorylation more efficiently in G2019S neurons than in R1441C neurons.
Conclusions:
- Human neurons with LRRK2 mutations exhibit genotype-dependent responses to IFNγ.
- These findings highlight the interplay between LRRK2 mutations and immune signaling pathways in PD.
- The study provides a foundation for further research into neuronal vulnerability mechanisms in Parkinson's disease.
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