Redox proteomics analyses of the influence of co-expression of wild-type or mutated LRRK2 and Tau on C. elegans protein expression and oxidative modification: relevance to Parkinson disease

Abstract

Aims: The human LRRK2 gene has been identified as the most common causative gene of autosomal-dominantly inherited and idiopathic Parkinson disease (PD). The G2019S substitution is the most common mutation in LRRK2. The R1441C mutation also occurs in cases of familial PD, but is not as prevalent. Some cases of LRRK2-based PD exhibit Tau pathology, which suggests that alterations on LRRK2 activity affect the pathophysiology of Tau. To investigate how LRRK2 might affect Tau and the pathophysiology of PD, we generated lines of C. elegans expressing human LRRK2 [wild-type (WT) or mutated (G2019S or R1441C)] with and without V337M Tau. Expression and redox proteomics were used to identify the effects of LRRK2 (WT and mutant) on protein expression and oxidative modifications.

Results: Co-expression of WT LRRK2 and Tau led to increased expression of numerous proteins, including several 60S ribosomal proteins, mitochondrial proteins, and the V-type proton ATPase, which is associated with autophagy. C. elegans expressing mutant LRRK2 showed similar changes, but also showed increased protein oxidation and lipid peroxidation, the latter indexed as increased protein-bound 4-hydroxy-2-nonenal (HNE).

Innovation: Our study brings new knowledge about the possible alterations induced by LRRK2 (WT and mutated) and Tau interactions, suggesting the involvement of G2019S and R1441C in Tau-dependent neurodegenerative processes.

Conclusion: These results suggest that changes in LRRK2 expression or activity lead to corresponding changes in mitochondrial function, autophagy, and protein translation. These findings are discussed with reference to the pathophysiology of PD.

FIG. 1.

Bar graph of body trashes count in 30 sec in C. elegans transgenic and nontransgenic strains. Raw data are reported.

FIG. 2.

(A) PCR gel of tau and LRRK2 gene expression; (B) Expression of GFP in the pharyngeal muscles of a young adult transgenic C. elegans expressing Tau V337M. Fluorescence was used as a preliminary indicator of transgene expression. Image taken with a 20× objective.

FIG. 3.

Bar graph of total protein carbonyls and total protein-bound 3NT in C. elegans transgenic and nontransgenic strains. Each value was compared before to the nontransgenic strain and after to the Tau strain, set as 100%. The symbols represent significant differences with p value<0.05.

FIG. 4.

Bar graph of total protein-bound HNE in C. elegans transgenic and nontransgenic strains. Each value was compared before to nontransgenic strain and after to Tau strain, set as 100%. The symbols represent significant differences with p value<0.05.

FIG. 5.

Representative 2D-gel with all the proteins identified in the expression proteomics study. The proteins are represented with identification numbers followed by the letter “e.” Consult Table 1 A–G for results of comparisons among the seven groups. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Representative 2D-gel with all the proteins identified in the redox proteomics study. The proteins are represented with identification numbers followed by the letter “h.” Consult Table 1 A–G for results of comparisons among the seven groups. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 7.

Representative 2D Western blot with all the proteins identified with altered HNE levels using redox proteomics analysis. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars.)

FIG. 8.

Proteomics data validation experiments. Top: Western blot of expression levels and protein-bound HNE levels of V-type proton ATPase in transgenic and nontransgenic C. elegans strains. Bottom: Bar graphs of significant differences between the groups analyzed are shown.

FIG. 9.

Comparision of GFP intensity between vehicle, rapamycin (200 nM) and ridaforolimus (200 nM) without (A) and with (B) bafliomycin; *p<0.05, **p<0.01, ***p<0.001.

FIG. 10.

Age synchronized nematodes cultured with varied doses of ridaforolimus. (A) Movement (thrashing) assessed on day 1 of adulthood. **p<0.01; (B) movement (thrashing) assessed on day 1, 3, and 5 of adulthood. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).