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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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Frataxin is essential for division and survival of murine fibroblasts.A. Schematic representation of wild-type murine frataxin allele (Frda+), the loxP-flanked Frda exon 4 allele (FrdaL3) and the Cre-mediated deleted allele (FrdaL-). Primers used for genotyping are represented by arrows [7]. B. Genotyping on heterozygous FrdaL3/L- cells before (lane 3) and after (lanes 4 and 5) pEGFP-Cre transfection and sort. Adherent living cells display a Frda L3/L-, Cre genotype (lane 4) whereas floating cells are completely deleted for frataxin (FrdaL-/L-) (lane 5). Lane 2: positive controls of each PCR. Lane 1: negative control (no DNA). C. Genotyping of FrdaL3/L- clones after pEGFP-Cre transfection and sort. Results from one clone transfected with an empty vector (pSG5: FrdaL3/L-; empty, clone B7) and two clones overexpressing murine frataxin (pSG5-mFxn: FrdaL3/L-; mFxn, clones C10 and E2). First lane: no DNA (−). Second lane: positive controls of each PCR (+). D. Phase contrast microscopy on rescued FrdaL3/L- clones after pEGFP-Cre transfection and sort. pSG5-mFxn: normal morphology of a murine frataxin overexpressing clone (FrdaL3/L-; mFxn) and deleted for endogenous frataxin. pSG5: cells from a clone transfected with the empty vector (FrdaL3/L-; empty). Note the presence of many elongated or round unhealthy cells. Phase contrast (20× magnification), cells plated in 96-well plastic plates.
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pone-0006379-g001: Frataxin is essential for division and survival of murine fibroblasts.A. Schematic representation of wild-type murine frataxin allele (Frda+), the loxP-flanked Frda exon 4 allele (FrdaL3) and the Cre-mediated deleted allele (FrdaL-). Primers used for genotyping are represented by arrows [7]. B. Genotyping on heterozygous FrdaL3/L- cells before (lane 3) and after (lanes 4 and 5) pEGFP-Cre transfection and sort. Adherent living cells display a Frda L3/L-, Cre genotype (lane 4) whereas floating cells are completely deleted for frataxin (FrdaL-/L-) (lane 5). Lane 2: positive controls of each PCR. Lane 1: negative control (no DNA). C. Genotyping of FrdaL3/L- clones after pEGFP-Cre transfection and sort. Results from one clone transfected with an empty vector (pSG5: FrdaL3/L-; empty, clone B7) and two clones overexpressing murine frataxin (pSG5-mFxn: FrdaL3/L-; mFxn, clones C10 and E2). First lane: no DNA (−). Second lane: positive controls of each PCR (+). D. Phase contrast microscopy on rescued FrdaL3/L- clones after pEGFP-Cre transfection and sort. pSG5-mFxn: normal morphology of a murine frataxin overexpressing clone (FrdaL3/L-; mFxn) and deleted for endogenous frataxin. pSG5: cells from a clone transfected with the empty vector (FrdaL3/L-; empty). Note the presence of many elongated or round unhealthy cells. Phase contrast (20× magnification), cells plated in 96-well plastic plates.

Mentions: Since fibroblasts of patients with mutations directly affecting the respiratory chain are viable in cell culture, irrespective of viability in the animal, we sought to obtain a FRDA cell model by frataxin deletion in mouse fibroblasts. To this end, an immortalized fibroblast cell line compound heterozygous for the mouse frataxin conditional allele and the deleted allele (FrdaL3/L-) [7] (Fig. 1A) was transfected with a fluorescent recombinase (pEGFP-Cre) and sorted by FACS 48 hours after transfection. Most sorted cells stopped dividing after 2–3 days in culture, became round, to end up floating in the culture medium after 7–10 days in culture. The genotype of the total cellular population demonstrated that not all adherent cells were deleted for frataxin (Fig. 1B, lane 4), while the floating cells harvested from the supernatant presented a clear FrdaL-/L- genotype (Fig. 1B, lane 5) suggesting that complete frataxin deletion is lethal in fibroblast.


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)

Frataxin is essential for division and survival of murine fibroblasts.A. Schematic representation of wild-type murine frataxin allele (Frda+), the loxP-flanked Frda exon 4 allele (FrdaL3) and the Cre-mediated deleted allele (FrdaL-). Primers used for genotyping are represented by arrows [7]. B. Genotyping on heterozygous FrdaL3/L- cells before (lane 3) and after (lanes 4 and 5) pEGFP-Cre transfection and sort. Adherent living cells display a Frda L3/L-, Cre genotype (lane 4) whereas floating cells are completely deleted for frataxin (FrdaL-/L-) (lane 5). Lane 2: positive controls of each PCR. Lane 1: negative control (no DNA). C. Genotyping of FrdaL3/L- clones after pEGFP-Cre transfection and sort. Results from one clone transfected with an empty vector (pSG5: FrdaL3/L-; empty, clone B7) and two clones overexpressing murine frataxin (pSG5-mFxn: FrdaL3/L-; mFxn, clones C10 and E2). First lane: no DNA (−). Second lane: positive controls of each PCR (+). D. Phase contrast microscopy on rescued FrdaL3/L- clones after pEGFP-Cre transfection and sort. pSG5-mFxn: normal morphology of a murine frataxin overexpressing clone (FrdaL3/L-; mFxn) and deleted for endogenous frataxin. pSG5: cells from a clone transfected with the empty vector (FrdaL3/L-; empty). Note the presence of many elongated or round unhealthy cells. Phase contrast (20× magnification), cells plated in 96-well plastic plates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2710521&req=5

pone-0006379-g001: Frataxin is essential for division and survival of murine fibroblasts.A. Schematic representation of wild-type murine frataxin allele (Frda+), the loxP-flanked Frda exon 4 allele (FrdaL3) and the Cre-mediated deleted allele (FrdaL-). Primers used for genotyping are represented by arrows [7]. B. Genotyping on heterozygous FrdaL3/L- cells before (lane 3) and after (lanes 4 and 5) pEGFP-Cre transfection and sort. Adherent living cells display a Frda L3/L-, Cre genotype (lane 4) whereas floating cells are completely deleted for frataxin (FrdaL-/L-) (lane 5). Lane 2: positive controls of each PCR. Lane 1: negative control (no DNA). C. Genotyping of FrdaL3/L- clones after pEGFP-Cre transfection and sort. Results from one clone transfected with an empty vector (pSG5: FrdaL3/L-; empty, clone B7) and two clones overexpressing murine frataxin (pSG5-mFxn: FrdaL3/L-; mFxn, clones C10 and E2). First lane: no DNA (−). Second lane: positive controls of each PCR (+). D. Phase contrast microscopy on rescued FrdaL3/L- clones after pEGFP-Cre transfection and sort. pSG5-mFxn: normal morphology of a murine frataxin overexpressing clone (FrdaL3/L-; mFxn) and deleted for endogenous frataxin. pSG5: cells from a clone transfected with the empty vector (FrdaL3/L-; empty). Note the presence of many elongated or round unhealthy cells. Phase contrast (20× magnification), cells plated in 96-well plastic plates.
Mentions: Since fibroblasts of patients with mutations directly affecting the respiratory chain are viable in cell culture, irrespective of viability in the animal, we sought to obtain a FRDA cell model by frataxin deletion in mouse fibroblasts. To this end, an immortalized fibroblast cell line compound heterozygous for the mouse frataxin conditional allele and the deleted allele (FrdaL3/L-) [7] (Fig. 1A) was transfected with a fluorescent recombinase (pEGFP-Cre) and sorted by FACS 48 hours after transfection. Most sorted cells stopped dividing after 2–3 days in culture, became round, to end up floating in the culture medium after 7–10 days in culture. The genotype of the total cellular population demonstrated that not all adherent cells were deleted for frataxin (Fig. 1B, lane 4), while the floating cells harvested from the supernatant presented a clear FrdaL-/L- genotype (Fig. 1B, lane 5) suggesting that complete frataxin deletion is lethal in fibroblast.

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