Journal article
Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation
Journal of Neuroscience Research, Vol.92(4), pp.506-516
2013
Abstract
Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T.
Details
- Title
- Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation
- Authors/Creators
- I.D. Greene (Author/Creator) - The University of Western AustraliaF. Mastaglia (Author/Creator) - The University of Western AustraliaB.P. Meloni (Author/Creator) - The University of Western AustraliaK.A. West (Author/Creator) - The University of Western AustraliaJ. Chieng (Author/Creator) - The University of Western AustraliaC.J. Mitchell (Author/Creator) - The University of Western AustraliaW-P Gai (Author/Creator) - Flinders UniversityS. Boulos (Author/Creator) - The University of Western Australia
- Publication Details
- Journal of Neuroscience Research, Vol.92(4), pp.506-516
- Publisher
- John Wiley & Sons Inc
- Identifiers
- 991005542067707891
- Copyright
- © 2013 Wiley Periodicals, Inc.
- Murdoch Affiliation
- Institute for Immunology and Infectious Diseases
- Language
- English
- Resource Type
- Journal article
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- Collaboration types
- Domestic collaboration
- Citation topics
- 1 Clinical & Life Sciences
- 1.52 Neurodegenerative Diseases
- 1.52.67 Parkinson's Disease
- Web Of Science research areas
- Neurosciences
- ESI research areas
- Neuroscience & Behavior