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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 24, 2005 as doi:10.1096/fj.05-4212fje. |
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* Laboratory of Genetics and Molecular Medicine, Department of Genomics and Proteomics, Instituto de Biomedicina, CSIC, Valencia, Spain; and
Department of Zoology, University of Cambridge, Cambridge, UK
1Correspondence: Department of Genomics and Proteomics, Instituto de Biomedicina, CSIC, C/ Jaume Roig, 11, Valencia 46010, Spain. E-mail: fpalau{at}ibv.csic.es
SPECIFIC AIMS
Work on model systems has proven to be essential to understanding frataxin function and therefore the pathogenic basis of Friedreich ataxia (FRDA). The aim of this work was to determine the results of reproducing the diminution of frataxin function observed in FRDA patients in the nematode Caenorhabditis elegans. This is the first step in establishing a new animal model for the study of frataxin function, which also has great potential for testing drugs to treat Friedreich ataxia.
PRINCIPAL FINDINGS
1. Identification and characterization of frh-1, the C. elegans FRDA ortholog
We identified the C. elegans homologue of the human FRDA gene, which encodes frataxin, which we called frataxin homologue(1frh-1) (GenBank accession no. AY048153). To determine the mRNA structure, we amplified the frh-1 cDNA by RT-PCR. frh-1 is processed by trans-splicing and is expressed as a part of an operon comprised of 8 genes.
2. Frataxin-depleted worms show a pleiotropic phenotype
To obtain partial reduction of frataxin, we performed RNAi of frh-1 gene by injection of dsRNA into the gonads of hermaphrodite worms. The F1 progeny of injected worms [frh-1(RNAi)] showed several phenotypes: slow growth (Gro), lethargic behavior, egg laying defects (Egl), reduced brood size, abnormal pharyngeal pumping (Eat), altered defecation (Dec), and low levels of larval lethality. As a negative control, we induced RNAi for the cat gene [E. coli chloramphenicol acetyl transferase; cat(RNAi)].
3. Reduction of function of frataxin produces abnormalities in pharyngeal pumping, due to neuronal damage
Pharynx pumping in C. elegans is a physiological event controlled by the nervous system. frh-1(RNAi) worms had significantly reduced pharyngeal pumping in the presence of food compared with the control cat(RNAi) (Student’s t test P<0.001). We also observed that pumping was arrhythmic. To further delimit which tissue, muscle or neurons, is affected, we cultured in the presence of serotonin. When the pharyngeal muscle function is intact serotonin is able to increase pumping of animals in the absence of food. When 7.5 mM serotonin was added to worms in the absence of food the pharynx pumping of the frh-1(RNAi) worms was increased to a higher rate than that measured in the frh-1(RNAi) worms in the presence of food. This result suggests that the main problem in the frataxin defective worms is due to a misfunction of the neurons that regulate pharyngeal pumping, although pharyngeal muscle function may also be compromised.
4. Frataxin is required for normal defecation in C. elegans
The defecation motor program (DMP) is initiated every 45 s and is controlled by the nervous system and by cyclic Ca2+ oscillations in the gut cells. We compared defecation behavior in the frh-1(RNAi) animals with both wild-type N2 and control cat(RNAi) worms. We observed that frh-1(RNAi) individuals had a significantly increased mean period between posterior body wall muscle (pBoc) contractions (meancat(RNAi)=60 s and meanfrh-1(RNAi)=92 s) (Student’s t test P<0.03). In addition, frh-1(RNAi) animals show a greater variability in the period of pBoc. Both of these observations suggest a misregulation of the defecation oscillator.
5. frh-1(RNAi) worms have increased sensitivity to oxidative stress and reduced life span
We performed experiments in which the RNAi worms were subjected to pro-oxidative stress environments. Some C. elegans mutants that have an increased sensitivity to oxidative stress are more susceptible to paraquat (methyl viologen), so we tested the effect of paraquat on frh-1(RNAi) animals. As a positive control we used the mev-1(kn1) strain, which shows high sensitivity to paraquat. The mev-1 gene encodes MEV-1, the C. elegans ortholog of human succinate dehydrogenase subunit C (SDHC), which is located in complex II of the mitochondrial electron transport chain. mev-1 mutants have an increased superoxide anion production and as a consequence age precociously. We incubated frh-1(RNAi), cat(RNAi) and mev-1(kn1) adult hermaphrodites in plates containing 0, 5, and 10 mM paraquat, respectively, at 25°C for 3 days, and assessed survival time. On the control plates, without paraquat, no deaths were observed during the course of the experiment. In contrast at 5 and 10 mM there was a significant increase (P<0.008 and P<0.0001, respectively) of deaths in frh-1(RNAi) worms compared with the cat(RNAi) controls (Fig. 1
A). mev-1(kn1) animals showed similar levels of sensitivity.
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To assess how this increased sensitivity to oxidative stress would affect the lifetime of the frataxin-deficient worms, we performed a life span assay on frh-1(RNAi) animals. We tested the lifetime of frh-1(RNAi) L4 larvae, and we used cat(RNAi) and mev-1(kn1) L4 worms as negative and positive control, respectively. Vitality was assessed by the response of animals to gentle nose touch. The life span was reduced in both frh-1(RNAi) worms (half-life 10 days) and mev-1(kn1) animals (half-life of 7.5 days). Differences were statistically significant (P<0.0001) when compared with the cat(RNAi) control worms (half-life 17 days) (Fig. 1B
). It is likely that decrease of life span of frh-1(RNAi) worms, due to reduced frataxin function, is related to the increased sensitivity to oxidative stress.
6. frh-1 and mev-1 interact functionally
We performed RNAi against the frh-1 gene in both wild-type and mev-1 mutant backgrounds, and we assessed whether there was some enhancement of the phenotype of the mev-1(kn1) worms. These two genes show a synergistic phenotype. We observed 44% embryonic lethality in the frh-1(RNAi)/mev-1(kn1) double knockdown mutant worms. This proportion was significantly different than wild-type, cat(RNAi), mev-1(kn1) and cat(RNAi)/mev-1(kn1) worms, all of which had very low levels (<5%) of embryonic lethality (P<0.0001) (Fig. 2
). The embryos that fully developed progressed beyond L1/L2. Many of them showed some early gonad structures, but all of them were sterile. These findings suggest that frh-1 and mev-1 interact genetically and indicate that frh-1 may have a role on the modulation of the complex II of the mitochondrial electron transport chain in C. elegans.
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7. frh-1 has a complex expression pattern in C. elegans
To know where frh-1 is expressed in C. elegans we transformed N2 worms with constructs carrying different genomic sequences of the frataxin gene fused to gfp. We found that there is a promoter of the frh-1 gene in its 5 kb region upstream, and regulatory sequences along this upstream region and in the gene itself, which modulates the expression of frh-1 in different cells. The FRH-1:GFP fusion protein was expressed in the pharynx, body wall muscles, gut, spermatheca, and in sensory neurons of the head.
CONCLUSIONS AND SIGNIFICANCE
We have shown that depletion of frataxin function in C. elegans causes an increased sensitivity to oxidative stress. We have demonstrated that frataxin-deficient worms age prematurely, likely due to the reported increased oxidative stress. We demonstrate that frh-1 interacts genetically with mev-1, which shows that frataxin may be necessary for the succinate dehydrogenase function. We show that depletion of frataxin causes abnormal pharyngeal pumping and misregulation of defecation.
As described above, our aim is to develop a C. elegans model to study frataxin and Friedreich ataxia. The expression pattern of the frh-1 gene shows that frataxin is needed in tissues with a high requirement of energy, like the pharynx and nervous system. These tissues are analogs of heart and human nervous system, where the human frataxin is highly expressed. Both tissues are affected in FRDA patients as they are in frataxin-deficient worms. Our results on oxidative stress and life span agreement with reported data in FRDA patients or model organisms for Friedreich ataxia. All these data show that C. elegans can be used as a model for the FRDA pathology, as it is possible to reproduce its particular characteristics. We also describe a new role of frataxin in modulation of the succinate dehydrogenase function. We propose that the frataxin is needed for an appropriate function of the succinate dehydrogenase, so when frataxin activity is reduced it leads to a misregulation of the complex II that may cause a leak of electrons and, subsequently, an oxidative stress problem and an abnormal oxidative phosphorylation. These two problems in turn can explain the reduction of life span and the altered defecation and pharyngeal pumping (Fig. 3
).
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We show the characteristics of a model of frataxin deficiency in the nematode C. elegans and propose that frataxin has a relevant role in the function of mitochondrial respiratory chain. Combined with other experimental systems, this model will provide insight into the molecular mechanisms underlying frataxin deficiency, and thus Friedreich ataxia, and be a useful tool for drug screening.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4212fje;
This article has been cited by other articles:
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