Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth

Abstract

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canal-associated neurons. We demonstrate that in the absence of lrk-1, the C. elegans homologue of human LRRK2, all phenotypic aspects of pink-1 loss-of-function mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background. These results provide the first evidence of an antagonistic role of PINK-1 and LRK-1. Due to the similarity of the C. elegans proteins to human LRRK2 and PINK1, we suggest a common role of both factors in cellular functions including stress response and regulation of neurite outgrowth. This study might help to link pink-1/PINK1 and lrk-1/LRRK2 function to the pathological processes resulting from Parkinson disease-related mutants in both genes, the first manifestations of which are cytoskeletal defects in affected neurons.

FIGURE 1.

pink-1 is ubiquitously expressed in C. elegans. The expression pattern of pink-1 was investigated by transgenic expression of a rescuing P_pink-1::pink-1::gfp [pink-1] construct in two independent pha-1 lines (BR3645 and BR3646). pink-1 is ubiquitously, but weakly expressed in all body regions including head and tail neurons (A and E), the pharynx (A), the distal tip cells (arrowheads), the CAN (open arrowheads), and vulva epithelium (asterisk). A and B, head region (head neurons, pharyngeal muscles). C and D, mid-body region (vulva epithelium). E and F, tail region (tail neurons). G and H, mid-body region (distal tip cell, CAN). The corresponding DIC pictures are shown (B, D, F, and H). I–K, co-localization of PINK-1-GFP (I) with Mitotracker Red (J) in the mitochondria (arrows) is shown in a merged image (K). Scale bars represent 20 (A–H) and 10 μm (I–K).

FIGURE 2.

The pink-1(tm1779)-induced oxidative stress sensitivity is suppressed by lrk-1 alleles tm1898 and km41 (A) and rescued by a wild type copy of pink-1 (B). Synchronized L4 larval animals were grown at 20 °C on NGM plates containing 150 mm paraquat and the survivors were counted 3 days later. Shown are mean values ± S.E. The percentage of survivors was normalized to wild type (wt). The total numbers of animals analyzed from individual strains are listed above each column (n). p values were calculated by t test analysis. **, p < 0.0017; and ***, p < 0.0007.

FIGURE 3.

lrk-1(tm1898) suppresses mitochondrial cristae defects of pink-1(tm1779). A, cross (top) and longitudinal (below) sections of mitochondria in C. elegans muscle cells. B, cross-sections of mitochondria in C. elegans neurons. C, quantitative analysis of cristae membrane length in muscle cells with respect to total mitochondrial area (μm²). D, cristae membrane length in neurons/μm² mitochondrium area. Shown are mean ± S.E. The number of analyzed mitochondrial profiles from at least 19 (in muscle cells) and 17 (in neurons), derived from at least two independent animals is listed above each column (n). p values were calculated by analysis of variance. **, p < 0.0050 and ***, p < 0.0001.

FIGURE 4.

Loss of pink-1 suppresses the tunicamycin sensitivity of lrk-1 mutants. The sensitivity of two lrk-1 alleles, tm1898 (A) and km41 (B) to treatment with 1.5 μg/ml tunicamycin resulted in a reduced survival rate of the animals that is suppressed by pink-1(tm1779). The total numbers of animals analyzed from individual strains are listed above each column (n). The percentage of survivors was normalized to wild type (wt). Shown are mean ± S.E. The distinct survival rate of pink-1(tm1779) animals between (A) and (B) is caused by the use of different tunicamycin batches for each set of experiments. Data of experiments obtained for B were carried out with strains harboring an additional integrated transgene ceh-10::gfp (indicated, for simplicity, as wild type (wt*)). **, p = 0.0030 and ***, p < 0.0003.

FIGURE 5.

The CAN axon pathfinding defects of pink-1 mutants are suppressed by loss of lrk-1 function. CAN axon pathfinding was visualized using the integrated transgene lqIs4[ceh-10::gfp] (20). Shown is the percentage (mean ± S.E.) of posterior CAN axons that terminated prematurely, were misguided, or branched inappropriately. A, both lrk-1 alleles tm1898 and km41 suppress CAN axon pathfinding defects of pink-1(tm1779). B, the CAN axon pathfinding defects of pink-1(tm1779) is rescued by transgenic expression of extrachromosomal pink-1::gfp. C, a genomic copy of lrk-1::gfp restores CAN axon pathfinding defects in lrk-1(tm1898);pink-1(tm1779) double mutants and, thus, suppresses the lrk-1(tm1898) phenotype. D, a genomic copy of lrk-1 (cosmid T27C10; byEx737–739) as well as a dominant version of LRK-1(G1876S) (lrk-1(tm1898);byEx734–736) leads to CAN axon pathfinding defects in both lrk-1(tm1898) mutant and in a wild type (wt) background. Dotted line in B–D, the mean value of pathfinding defects in wild type (wt) animals carrying the lqIs4-integrated ceh-10::gfp transgene is shown. The total numbers of animals analyzed from individual strains are listed above each column (n). *, p = 0.0295; **, p < 0.0064; and ***, p < 0.0007.