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.

Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease

“…Fluorescence imaging of transfected HEK293 cells confirmed LRRK2 expression at 48 and 72 hours post-transfection (Figure 1(b)). consistent with our previous study, GFP levels appeared to be reduced in R1441C and A1442P transfected HEK293 cells (Figure 1(b)) [5].…”

“…The primer pairs were as follows: To confirm the site directed mutagenesis, the resultant constructs were fully sequenced. The other plasmid constructs used in this study are previously described [5].…”

“…We previously reported that the ROC domain missense mutation A1442P, identified in a Western Australian patient, caused reduced LRRK2 steady state protein levels, as did the common R1441C mutation [5]. Mutations within the ROC domain of LRRK2 reduce GTP hydrolysis and LRRK2 protein autophosphorylation, which leads to dysregulated organelle degradation, altered signalling and ultimately immune dysfunction [1] [6] [7].…”

“…fused with the N-terminus green fluorescent protein (GFP) sequence and FLAG peptide sequence has previously been described [5]. The pcDNA3.1 Thr2397 LRRK2 construct was used as a template for site-directed mutagenesis to produce the Met2397 LRRK2 haplotype.…”

“…The LRRK2 Met2397 haplotype was derived by site directed mutagenesis as previously described [5], using a plasmid pCDNA3.1 encoding the GFP-LRRK2-FLAG Thr2397 haplotype as the template. The primer pairs were as follows: To confirm the site directed mutagenesis, the resultant constructs were fully sequenced.…”