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Assaying the Kinase Activity of LRRK2 in vitro
Published on: January 18, 2012
LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity
Hye Young Heo1, Ji-Min Park, Cy-Hyun Kim
1Institute for Brain Science and Technology, Busan, South Korea.
Abstract:
LRRK2 is an autosomal dominant gene whose mutations cause familial Parkinson's disease (PD). The LRRK2 protein contains a functional kinase and a GTPase domain. PD phenotypes caused by LRRK2 mutations are similar to those of idiopathic PD, implying that LRRK2 is an important participant in PD pathogenesis. Of LRRK2's PD-specific mutations, the G2019S is the most frequently observed one. Its over-expression is known to increase kinase activity and neurotoxicity compared to wild type (WT) LRRK2. Here, using a simple colorimetric cell viability assay, we analyzed LRRK2's neurotoxicity in dopaminergic SN4741 cells following treatment with hydrogen peroxide. When WT, G2019S, or empty vector was expressed in SN4741 cells, cell death was modestly and significantly increased in the order of G2019S>WT>vector. When these transfected cells were treated with hydrogen peroxide to mimic oxidative stress, cellular neurotoxicity was enhanced in the same order (i.e. G2019S>WT>vector). Moreover, incubation of SN4741 cells with conditioned medium from cells expressing G2019S and subjected to hydrogen peroxide treatment exhibited 10-15% more cell death than conditioned medium from cells transfected with vector or WT, suggesting that G2019S-expressing cells secrete a factor(s) affecting viability of neighboring cells. The kinase domain was mapped to be responsible for oxidative stress-induced neurotoxicity. In addition, over-expression of WT and G2019S LRRK2 lead to a weak, but significant, increase in intracellular reactive oxygen species (ROS) in the order of G2019S>WT as measured by DCFH-DA assay in both the presence and absence of H(2)O(2) treatment. Furthermore, in G2019S-expressing cells, co-expression of the anti-oxidant protein DJ-1 or ERK inhibitor treatment restored survival rate to a level similar to that of cells transfected with control vector under H(2)O(2) treatment. Taken together, our data suggest that the LRRK2 kinase domain increases the generation of ROS and causes enhanced neurotoxicity under H(2)O(2) treatment, which can be at least partially rescued by DJ-1 or the ERK inhibitor.
Insights
Mutant LRRK2, particularly the G2019S variant, increases oxidative stress and neurotoxicity in Parkinson's disease models. This effect, mediated by the kinase domain and reactive oxygen species, can be partially reversed by DJ-1 or ERK inhibition.
Area of Science:
- Neuroscience
- Genetics
- Cell Biology
Background:
- Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD).
- The G2019S mutation is the most prevalent PD-associated LRRK2 variant, known to enhance kinase activity and neurotoxicity.
- LRRK2's role in PD pathogenesis is significant, with its dysfunction mirroring idiopathic PD phenotypes.
Purpose of the Study:
- To investigate the neurotoxicity of wild-type (WT) and G2019S mutant LRRK2 in dopaminergic cells under oxidative stress.
- To elucidate the mechanisms underlying LRRK2-mediated neurotoxicity, including the role of reactive oxygen species (ROS) and specific protein domains.
- To explore potential therapeutic interventions for LRRK2-associated neurotoxicity.
Main Methods:
- Utilized a colorimetric cell viability assay to assess neurotoxicity in SN4741 dopaminergic cells expressing WT LRRK2, G2019S LRRK2, or an empty vector.
- Induced oxidative stress using hydrogen peroxide (H2O2) treatment.
- Measured intracellular reactive oxygen species (ROS) levels using the DCFH-DA assay.
- Investigated the effect of conditioned medium from transfected cells on neighboring cell viability.
- Examined the impact of DJ-1 co-expression and ERK inhibitor treatment on cell survival.
Main Results:
- Over-expression of G2019S LRRK2 led to significantly increased cell death compared to WT LRRK2 and vector controls, both with and without H2O2 treatment.
- LRRK2 expression, particularly the G2019S mutant, enhanced neurotoxicity under oxidative stress conditions.
- Conditioned medium from G2019S-expressing cells induced higher cell death, suggesting paracrine signaling.
- The kinase domain of LRRK2 was identified as crucial for oxidative stress-induced neurotoxicity.
- LRRK2 expression increased intracellular ROS levels, with G2019S showing a greater effect than WT.
- DJ-1 co-expression and ERK inhibition partially rescued cell survival in G2019S-expressing cells under oxidative stress.
Conclusions:
- The LRRK2 kinase domain contributes to enhanced neurotoxicity and ROS generation under oxidative stress, a key factor in Parkinson's disease pathogenesis.
- LRRK2 mutations may exert toxic effects through both intrinsic cellular mechanisms and by influencing neighboring cells.
- DJ-1 and ERK pathway modulation represent potential therapeutic strategies for LRRK2-associated Parkinson's disease.
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