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. 2009 Jul 22;29(29):9210-8.
doi: 10.1523/JNEUROSCI.2281-09.2009.

LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans

Shamol Saha  1 , Maria D Guillily, Andrew Ferree, Joel Lanceta, Diane Chan, Joy Ghosh, Cindy H Hsu, Lilach Segal, Kesav Raghavan, Kunihiro Matsumoto, Naoki Hisamoto, Tomoki Kuwahara, Takeshi Iwatsubo, Landon Moore, Lee Goldstein, Mark Cookson, Benjamin Wolozin
Affiliations

LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans

Shamol Saha et al. J Neurosci. .

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

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Figures

Figure 1.
Figure 1.
Generation and characterization of C. elegans lines expressing WT or G2019S LRRK2. A, Quantification of LRRK2 mRNA expression for the WT LRRK2 lines (wlzIs1 and wlzIs2) and G2019S LRRK2 lines (wlzIs3 and wlzIs4). LRRK2 expression level was only significantly different for line wlzIs1 (*p < 0.001). B, Expression of LRRK2 in C. elegans lines. Three successive fractions eluted from the anti-V5 antibody column are shown for each nematode line. Lanes 1–3, WT LRRK2 (line wlzIs2); lanes 4–6, Mec-4::GFP (a line derived from the same lin-15 background as the LRRK2 lines); lanes 7–9, G2019S LRRK2 (line wlzIs4). The arrow points to the V5-LRRK2 at 250 kDa, which was eluted from the anti-V5 column. C, Number of eggs laid by N2 and transgenic lines. D, Analysis of total brood size for WT and G2019S LRRK2 (lines wlzIs2 and wlzIs4) demonstrates similar levels of fertility. E, F, Analysis of lifespan for WT and G2019S LRRK2 shows that C. elegans expressing WT LRRK2 (lines wlzIs1, wlzIs2) exhibit greater median lifespan compared with nontransgenic (N2) or G2019S LRRK2 lines (lines wlzIs3, wlzIs4). The lifespan data for E and F were generated with separate experiments.
Figure 2.
Figure 2.
LRRK2 protects against mitochondrial stress. A, Nematode expressing WT or G2019S LRRK2 (lines wlzIs2 and wlzIs4) showed reduced toxicity to rotenone (25 μm). B, Nematodes expressing WT LRRK2 (strain wlzIs2) showed reduced toxicity to paraquat (500 μm). The G2019S LRRK2 strain (strain wlzIs4) showed sensitivity to paraquat similar to that of N2. The p values represent overall p values for each figure. WT and G2019S LRRK2 were significantly different (p < 0.0001) in A and B; N2 and G2019S LRRK2 were not significantly different in B. C, R1441C, G2019S, and KD LRRK2 (wlzls5, wlzls4, and wlzIs7) showed less protection against rotenone (10 μm) than WT LRRKS (wlzls2), and KD/R1441C LRRK2 (wlzls6) was strongly protected against rotenone (20 μm) N2 (n = 12, 40 nematodes per dish for all points). D, Bristol N2 and lin-15 (the background strain for used for producing the transgenic C. elegans lines) showed similar levels of sensitivity to rotenone (25 μm, 4 d).
Figure 3.
Figure 3.
Endogenous C. elegans lrk-1 modulates the response to mitochondrial stress. A, Knockdown of lrk-1 enhanced rotenone (25 μm) toxicity of Bristol N2 C. elegans. B, The lrk-1 [km17] line, lacking the lrk-1 kinase and WD40 domains, showed reduced protection against rotenone compared with the control Bristol N2 line. C, WT LRRK2 (wlzsIs2), but not G2019S (wlzsIs4), partially complemented the lrk-1 [km17] deletion after 4 d on 10 μm rotenone (data normalized to the parent line).
Figure 4.
Figure 4.
LRRK2 induces loss of DAT::GFP fluorescence in dopaminergic neurons of adult nematodes. A, DAT::GFP fluorescence at different ages. The arrows point to the CEP dopaminergic neurons. The LRRK2 lines showed an accelerated decrease in DAT::GFP signal. B, Quantification of fluorescence signal from the nematode lines beginning with larval stage 3 and continuing on in days of adult life. C, High magnification (100×) showing representative DAT::GFP fluorescence in the WT and G2019S LRRK2 lines at day 2. D, G2019S LRRK2 (line wlzIs4) shows reduced dopamine levels at day 2 of adult life compared with the WT LRRK2 (line wlzIs2) or nontransgenic (N2) line (*p < 0.05).
Figure 5.
Figure 5.
G2019S LRRK2 enhances toxicity in dopaminergic neurons to mitochondrial dysfunction. A, Fluorescence of DAT::GFP (a), WT LRRK2/DAT::GFP (b), and G2019S LRRK2/DAT::GFP (c) lines during exposure to rotenone (250 nm, 24 h). Arrows point to CEP dopaminergic neurons in the neural ring. B, Quantification of fluorescence from dopaminergic neurons in each nematode line. C, Quantification of dopamine in each nematode line at adult day 3 with or without 250 nm rotenone.
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