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The first cellular models based on frataxin missense mutations that reproduce spontaneously the defects associated with Friedreich ataxia.

Calmels N, Schmucker S, Wattenhofer-Donzé M, Martelli A, Vaucamps N, Reutenauer L, Messaddeq N, Bouton C, Koenig M, Puccio H - PLoS ONE (2009)

Bottom Line: This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin.Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype.Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

ABSTRACT

Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.

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Biochemical measurements of ISC enzyme activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. Complex II of respiratory chain (Succinate Quinone DCPIP Reductase, SQDR; white bars) and aconitases (grey bars) specific activities were standardized to isocitrate dehydrogenase (IDH) specific activity and expressed as percentage of control activity. B. IRP1/IRE binding activity was measured by EMSA. Signals in the absence and in the presence of 2% of 2-βmercaptoethanol (βME) were quantified to determine the percentage of IRE-binding IRP1 in each clone. C. Nth1 activity was determined by the thymidine glycol nicking activity of crude nuclear extracts of each clone. Results, expressed as percentage of control activity, are given as the mean of the quantified intensity of Nth1 cleaved product signal from the thymidine glycol containing oligo observed on a 20% denaturing polyacrylamide gel. D. Western blot analysis using a specific anti-GPAT antibody. GAPDH was used as a loading control. GPAT C1F represents COS cells expressing a mutated GPAT which cannot be maturated, giving the GPAT precursor migration size. All values were generated by determining the means of two or three independent experiments performed on different cell extracts on at least 3 clones of hFXN, hFXNG130V or hFXNI154F. Data are represented as mean + SD. * p<0.05; *** p<0.005.
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pone-0006379-g005: Biochemical measurements of ISC enzyme activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. Complex II of respiratory chain (Succinate Quinone DCPIP Reductase, SQDR; white bars) and aconitases (grey bars) specific activities were standardized to isocitrate dehydrogenase (IDH) specific activity and expressed as percentage of control activity. B. IRP1/IRE binding activity was measured by EMSA. Signals in the absence and in the presence of 2% of 2-βmercaptoethanol (βME) were quantified to determine the percentage of IRE-binding IRP1 in each clone. C. Nth1 activity was determined by the thymidine glycol nicking activity of crude nuclear extracts of each clone. Results, expressed as percentage of control activity, are given as the mean of the quantified intensity of Nth1 cleaved product signal from the thymidine glycol containing oligo observed on a 20% denaturing polyacrylamide gel. D. Western blot analysis using a specific anti-GPAT antibody. GAPDH was used as a loading control. GPAT C1F represents COS cells expressing a mutated GPAT which cannot be maturated, giving the GPAT precursor migration size. All values were generated by determining the means of two or three independent experiments performed on different cell extracts on at least 3 clones of hFXN, hFXNG130V or hFXNI154F. Data are represented as mean + SD. * p<0.05; *** p<0.005.

Mentions: To assess the consequences of the missense mutations on the ISC-related function of frataxin, the activities of mitochondrial, cytosolic and nuclear ISC containing enzymes known to be decreased in FRDA heart biopsies [5] or FRDA conditional mouse models [7]–[9] were measured. Both hFXNG130V and hFXNI154F clones showed a significant decrease in the mitochondrial succinate dehydrogenase (SDH) activity with a more severe effect in the hFXNI154F mutants compared to the hFXNG130V mutants with 36% and 54% residual SDH activity, respectively (Fig. 5A). The activity of the aconitases was also decreased in both hFXNG130V and hFXNI154F clones, although to a lesser degree than the SDH activity, with 74% and 64% residual aconitase activity, respectively (Fig. 5A). The iron regulatory protein 1 (IRP1) is a bifunctional ISC protein located in the cytosol [31]. In the presence of [4Fe-4S] cluster, it functions as an aconitase. Devoid of its ISC, it binds to specific mRNA stem loop structures called IRE to regulate the expression of proteins involved in iron homeostasis. An increase in the IRE binding activity of IRP1 was measured only in the hFXNI154F clones compared to the parental cells (Fig. 5B). To determine whether there was a deficit in a nuclear ISC protein, we evaluated the activity of Nth1, a [4Fe-4S] glycosylase/AP-lyase involved in the base excision repair of oxidized bases such as thymine glycol (Tg) [32]. A significant decrease in the activity of Nth1was observed specifically in the hFXNI154F clones (Fig. 5C). The change in activity was not due to a transcriptional regulation, as RT-PCR showed no difference in Nth1 expression between the different clones (data not shown). Note that the decrease in the ISC enzymes activities described above occurs spontaneously, without the addition of exogenous stress. Furthermore, the severity of the deficit was variable from one clone to another. Finally, not all ISC enzymes were affected in the hFXNI154F clones. Indeed, glutamine phosphoribosylpyrophosphate aminotranferase (GPAT), an enzyme of purine biosynthesis which requires the incorporation of a [4Fe-4S] cluster to generate the mature enzyme [33], is not affected in both the hFXNG130V and hFXNI154F clones (Fig. 5D), suggesting that some ISC enzymes are less sensitive to frataxin functional impairment.


The first cellular models based on frataxin missense mutations that reproduce spontaneously the defects associated with Friedreich ataxia.

Calmels N, Schmucker S, Wattenhofer-Donzé M, Martelli A, Vaucamps N, Reutenauer L, Messaddeq N, Bouton C, Koenig M, Puccio H - PLoS ONE (2009)

Biochemical measurements of ISC enzyme activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. Complex II of respiratory chain (Succinate Quinone DCPIP Reductase, SQDR; white bars) and aconitases (grey bars) specific activities were standardized to isocitrate dehydrogenase (IDH) specific activity and expressed as percentage of control activity. B. IRP1/IRE binding activity was measured by EMSA. Signals in the absence and in the presence of 2% of 2-βmercaptoethanol (βME) were quantified to determine the percentage of IRE-binding IRP1 in each clone. C. Nth1 activity was determined by the thymidine glycol nicking activity of crude nuclear extracts of each clone. Results, expressed as percentage of control activity, are given as the mean of the quantified intensity of Nth1 cleaved product signal from the thymidine glycol containing oligo observed on a 20% denaturing polyacrylamide gel. D. Western blot analysis using a specific anti-GPAT antibody. GAPDH was used as a loading control. GPAT C1F represents COS cells expressing a mutated GPAT which cannot be maturated, giving the GPAT precursor migration size. All values were generated by determining the means of two or three independent experiments performed on different cell extracts on at least 3 clones of hFXN, hFXNG130V or hFXNI154F. Data are represented as mean + SD. * p<0.05; *** p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2710521&req=5

pone-0006379-g005: Biochemical measurements of ISC enzyme activities in wild-type hFXN, mutant hFXNG130V and hFXNI154F clones.A. Complex II of respiratory chain (Succinate Quinone DCPIP Reductase, SQDR; white bars) and aconitases (grey bars) specific activities were standardized to isocitrate dehydrogenase (IDH) specific activity and expressed as percentage of control activity. B. IRP1/IRE binding activity was measured by EMSA. Signals in the absence and in the presence of 2% of 2-βmercaptoethanol (βME) were quantified to determine the percentage of IRE-binding IRP1 in each clone. C. Nth1 activity was determined by the thymidine glycol nicking activity of crude nuclear extracts of each clone. Results, expressed as percentage of control activity, are given as the mean of the quantified intensity of Nth1 cleaved product signal from the thymidine glycol containing oligo observed on a 20% denaturing polyacrylamide gel. D. Western blot analysis using a specific anti-GPAT antibody. GAPDH was used as a loading control. GPAT C1F represents COS cells expressing a mutated GPAT which cannot be maturated, giving the GPAT precursor migration size. All values were generated by determining the means of two or three independent experiments performed on different cell extracts on at least 3 clones of hFXN, hFXNG130V or hFXNI154F. Data are represented as mean + SD. * p<0.05; *** p<0.005.
Mentions: To assess the consequences of the missense mutations on the ISC-related function of frataxin, the activities of mitochondrial, cytosolic and nuclear ISC containing enzymes known to be decreased in FRDA heart biopsies [5] or FRDA conditional mouse models [7]–[9] were measured. Both hFXNG130V and hFXNI154F clones showed a significant decrease in the mitochondrial succinate dehydrogenase (SDH) activity with a more severe effect in the hFXNI154F mutants compared to the hFXNG130V mutants with 36% and 54% residual SDH activity, respectively (Fig. 5A). The activity of the aconitases was also decreased in both hFXNG130V and hFXNI154F clones, although to a lesser degree than the SDH activity, with 74% and 64% residual aconitase activity, respectively (Fig. 5A). The iron regulatory protein 1 (IRP1) is a bifunctional ISC protein located in the cytosol [31]. In the presence of [4Fe-4S] cluster, it functions as an aconitase. Devoid of its ISC, it binds to specific mRNA stem loop structures called IRE to regulate the expression of proteins involved in iron homeostasis. An increase in the IRE binding activity of IRP1 was measured only in the hFXNI154F clones compared to the parental cells (Fig. 5B). To determine whether there was a deficit in a nuclear ISC protein, we evaluated the activity of Nth1, a [4Fe-4S] glycosylase/AP-lyase involved in the base excision repair of oxidized bases such as thymine glycol (Tg) [32]. A significant decrease in the activity of Nth1was observed specifically in the hFXNI154F clones (Fig. 5C). The change in activity was not due to a transcriptional regulation, as RT-PCR showed no difference in Nth1 expression between the different clones (data not shown). Note that the decrease in the ISC enzymes activities described above occurs spontaneously, without the addition of exogenous stress. Furthermore, the severity of the deficit was variable from one clone to another. Finally, not all ISC enzymes were affected in the hFXNI154F clones. Indeed, glutamine phosphoribosylpyrophosphate aminotranferase (GPAT), an enzyme of purine biosynthesis which requires the incorporation of a [4Fe-4S] cluster to generate the mature enzyme [33], is not affected in both the hFXNG130V and hFXNI154F clones (Fig. 5D), suggesting that some ISC enzymes are less sensitive to frataxin functional impairment.

Bottom Line: This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin.Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype.Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

ABSTRACT

Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.

Show MeSH
Related in: MedlinePlus