title: Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation.
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.
authors.affiliation: Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Australian Neuro-Muscular Research Institute, Nedlands, Western Australia, Australia.
authors.forename: Izabella D
authors.initials: ID
authors.lastname: Greene
authors.forename: Francis
authors.initials: F
authors.lastname: Mastaglia
authors.forename: Bruno P
authors.initials: BP
authors.lastname: Meloni
authors.forename: Kristin A
authors.initials: KA
authors.lastname: West
authors.forename: Joanne
authors.initials: J
authors.lastname: Chieng
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Mitchell
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Gai
authors.forename: Sherif
authors.initials: S
authors.lastname: Boulos
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000704:Analysis of Variance; D002470:Cell Survival; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D013997:Time Factors; D014162:Transfection
chemical_list: D000596:Amino Acids; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: A1442P; LRRK2; Parkinson's disease; R1441C; ROC domain mutations