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Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation

Authors

  • Izabella D. Greene,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
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  • Francis Mastaglia,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
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  • Bruno P. Meloni,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
    2. Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
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  • Kristin A. West,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
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  • Joanne Chieng,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
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  • Chris J. Mitchell,

    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
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  • Wei-Ping Gai,

    1. Department of Human Physiology and Centre for Neuroscience, Flinders University School of Medicine, Bedford Park, South Australia, Australia
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  • Sherif Boulos

    Corresponding author
    1. Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia
    • Correspondence to: Sherif Boulos, Australian Neuromuscular Research Institute, A Block, 4th Floor, QEII Medical Centre, Nedlands, Western Australia, Australia 6009. E-mail: sherif.boulos@anri.uwa.edu.au

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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. © 2013 Wiley Periodicals, Inc.

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