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The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease.

Niemann A, Huber N, Wagner KM, Somandin C, Horn M, Lebrun-Julien F, Angst B, Pereira JA, Halfter H, Welzl H, Feltri ML, Wrabetz L, Young P, Wessig C, Toyka KV, Suter U - Brain (2014)

Bottom Line: GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane.This translocation is necessary to substitute for loss of GDAP1 expression.We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

View Article: PubMed Central - PubMed

Affiliation: 1 Institute of Molecular Health Sciences, Cell Biology, Department of Biology, ETH Zurich, Swiss Federal Institute of Technology, Switzerland, ETH-Hönggerberg, 8093 Zürich, Switzerland.

ABSTRACT
The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. We found that Gdap1 knockout mice (Gdap1(-/-)), mimicking genetic alterations of patients suffering from severe forms of Charcot-Marie-Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1(-/-) mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1(-/-) mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1(-/-) mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1(-/-) mice compared with controls. Our findings demonstrate that Charcot-Marie-Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

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Mitochondrial DNA biogenesis is increased in sciatic nerves of Gdpa1−/− mice. (A) DNA of the indicated tissues was isolated from 19-month-old Gdap1−/− and wild-type mice. The relative amount of mitochondrial DNA (mtDNA) in relation to genomic DNA (gDNA) was determined by quantitative PCR. (B) Total RNA was isolated from sciatic nerve (SCN) and spinal cord (SPC) lysates from 19-month-old Gdap1−/− and wild-type mice. The relative expression levels of nuclear factors involved in mitochondrial biogenesis and redox protection were determined by quantitative PCR in relation to β-actin levels. (A and B) Graphs represent means and standard error of tissues of n = 3 animals per genotype, n.s. = not significant, *P < 0.05.
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awt371-F4: Mitochondrial DNA biogenesis is increased in sciatic nerves of Gdpa1−/− mice. (A) DNA of the indicated tissues was isolated from 19-month-old Gdap1−/− and wild-type mice. The relative amount of mitochondrial DNA (mtDNA) in relation to genomic DNA (gDNA) was determined by quantitative PCR. (B) Total RNA was isolated from sciatic nerve (SCN) and spinal cord (SPC) lysates from 19-month-old Gdap1−/− and wild-type mice. The relative expression levels of nuclear factors involved in mitochondrial biogenesis and redox protection were determined by quantitative PCR in relation to β-actin levels. (A and B) Graphs represent means and standard error of tissues of n = 3 animals per genotype, n.s. = not significant, *P < 0.05.

Mentions: Impairing mitochondrial dynamics influences the mitochondrial DNA content. Blocking mitochondrial fusion leads to a reduction or loss of mitochondrial DNA, which can be quantified by the ratio between mitochondrial DNA and genomic DNA (Chen and Chan, 2010; Malik and Czajka, 2013). Expression knockdown of the mitochondrial fission factor dynamin-related protein 1 (DRP1, now known as DNM1L) also reduces the mitochondrial DNA content (Parone et al., 2008), whereas in Drp1−/− mouse embryonic fibroblasts the mitochondrial DNA content was not significantly altered (Ishihara et al., 2009). As GDAP1 is a mitochondrial fission factor and mitochondrial morphology was altered in the plantar nerve of Gdap1−/− animals (Supplementary Fig. 2D), we determined the mitochondrial DNA content in relation to the genomic DNA. Unexpectedly, the mitochondrial to genomic DNA-ratio was significantly increased in the sciatic nerve of Gdap1−/− animals compared with age-matched controls (Fig. 4A). Increased mitochondrial DNA contents are characteristic for mild, persistent oxidative stress, which is not overloading the intracellular antioxidant system (Lee and Wei, 2005; Scarpulla et al., 2012). Nuclear factors have been identified to regulate this protective response (Lee and Wei, 2005; Moreno-Loshuertos et al., 2006; Malik and Czajka, 2013). The peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α, now known as PPARGC1A) regulates various aspects of mitochondrial biogenesis, including the protective mitochondrial DNA increase. PGC1α influences the mitochondrial DNA replication in concert with other nuclear factors and coactivators, mainly nuclear respiratory factors 1 and 2 (NRF1, NRF2) and oestrogen-related receptor α (ERRα, now known as ESRRA). The transcription factor A, mitochondrial (TFAM) is one of the factors being induced by PGC1α and its coactivators. As mitochondrial factor, TFAM positively regulates the mitochondrial DNA copy number. Additionally, PGC1α and ERRα induce sirtuin 3 (SIRT3), which activates reactive oxygen species-protective pathways in mitochondria, i.e. the mitochondrial superoxide dismutase 2 (SOD2; Lee and Wei, 2005; Scarpulla et al., 2012). We found that the messenger RNA levels of Pgc1α are significantly increased in the sciatic nerves of Gdap1−/− mice compared with controls (Fig. 4B). In addition Errα, Tfam and Sod2 were elevated although significance was not reached due to variability in Gdap1−/− samples. No changes in Sirt3 levels were detectable (Fig. 4B). Collectively, our findings support that mild, persistent oxidative stress leads to an increase in the mitochondrial DNA content in sciatic nerves of 19-month-old Gdap1−/− mice. Strikingly, none of these changes were detected in highly GDAP1 expressing CNS tissues (Niemann et al., 2005; Pedrola et al., 2005) (Fig. 4A and B).Figure 4


The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease.

Niemann A, Huber N, Wagner KM, Somandin C, Horn M, Lebrun-Julien F, Angst B, Pereira JA, Halfter H, Welzl H, Feltri ML, Wrabetz L, Young P, Wessig C, Toyka KV, Suter U - Brain (2014)

Mitochondrial DNA biogenesis is increased in sciatic nerves of Gdpa1−/− mice. (A) DNA of the indicated tissues was isolated from 19-month-old Gdap1−/− and wild-type mice. The relative amount of mitochondrial DNA (mtDNA) in relation to genomic DNA (gDNA) was determined by quantitative PCR. (B) Total RNA was isolated from sciatic nerve (SCN) and spinal cord (SPC) lysates from 19-month-old Gdap1−/− and wild-type mice. The relative expression levels of nuclear factors involved in mitochondrial biogenesis and redox protection were determined by quantitative PCR in relation to β-actin levels. (A and B) Graphs represent means and standard error of tissues of n = 3 animals per genotype, n.s. = not significant, *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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awt371-F4: Mitochondrial DNA biogenesis is increased in sciatic nerves of Gdpa1−/− mice. (A) DNA of the indicated tissues was isolated from 19-month-old Gdap1−/− and wild-type mice. The relative amount of mitochondrial DNA (mtDNA) in relation to genomic DNA (gDNA) was determined by quantitative PCR. (B) Total RNA was isolated from sciatic nerve (SCN) and spinal cord (SPC) lysates from 19-month-old Gdap1−/− and wild-type mice. The relative expression levels of nuclear factors involved in mitochondrial biogenesis and redox protection were determined by quantitative PCR in relation to β-actin levels. (A and B) Graphs represent means and standard error of tissues of n = 3 animals per genotype, n.s. = not significant, *P < 0.05.
Mentions: Impairing mitochondrial dynamics influences the mitochondrial DNA content. Blocking mitochondrial fusion leads to a reduction or loss of mitochondrial DNA, which can be quantified by the ratio between mitochondrial DNA and genomic DNA (Chen and Chan, 2010; Malik and Czajka, 2013). Expression knockdown of the mitochondrial fission factor dynamin-related protein 1 (DRP1, now known as DNM1L) also reduces the mitochondrial DNA content (Parone et al., 2008), whereas in Drp1−/− mouse embryonic fibroblasts the mitochondrial DNA content was not significantly altered (Ishihara et al., 2009). As GDAP1 is a mitochondrial fission factor and mitochondrial morphology was altered in the plantar nerve of Gdap1−/− animals (Supplementary Fig. 2D), we determined the mitochondrial DNA content in relation to the genomic DNA. Unexpectedly, the mitochondrial to genomic DNA-ratio was significantly increased in the sciatic nerve of Gdap1−/− animals compared with age-matched controls (Fig. 4A). Increased mitochondrial DNA contents are characteristic for mild, persistent oxidative stress, which is not overloading the intracellular antioxidant system (Lee and Wei, 2005; Scarpulla et al., 2012). Nuclear factors have been identified to regulate this protective response (Lee and Wei, 2005; Moreno-Loshuertos et al., 2006; Malik and Czajka, 2013). The peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α, now known as PPARGC1A) regulates various aspects of mitochondrial biogenesis, including the protective mitochondrial DNA increase. PGC1α influences the mitochondrial DNA replication in concert with other nuclear factors and coactivators, mainly nuclear respiratory factors 1 and 2 (NRF1, NRF2) and oestrogen-related receptor α (ERRα, now known as ESRRA). The transcription factor A, mitochondrial (TFAM) is one of the factors being induced by PGC1α and its coactivators. As mitochondrial factor, TFAM positively regulates the mitochondrial DNA copy number. Additionally, PGC1α and ERRα induce sirtuin 3 (SIRT3), which activates reactive oxygen species-protective pathways in mitochondria, i.e. the mitochondrial superoxide dismutase 2 (SOD2; Lee and Wei, 2005; Scarpulla et al., 2012). We found that the messenger RNA levels of Pgc1α are significantly increased in the sciatic nerves of Gdap1−/− mice compared with controls (Fig. 4B). In addition Errα, Tfam and Sod2 were elevated although significance was not reached due to variability in Gdap1−/− samples. No changes in Sirt3 levels were detectable (Fig. 4B). Collectively, our findings support that mild, persistent oxidative stress leads to an increase in the mitochondrial DNA content in sciatic nerves of 19-month-old Gdap1−/− mice. Strikingly, none of these changes were detected in highly GDAP1 expressing CNS tissues (Niemann et al., 2005; Pedrola et al., 2005) (Fig. 4A and B).Figure 4

Bottom Line: GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane.This translocation is necessary to substitute for loss of GDAP1 expression.We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

View Article: PubMed Central - PubMed

Affiliation: 1 Institute of Molecular Health Sciences, Cell Biology, Department of Biology, ETH Zurich, Swiss Federal Institute of Technology, Switzerland, ETH-Hönggerberg, 8093 Zürich, Switzerland.

ABSTRACT
The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. We found that Gdap1 knockout mice (Gdap1(-/-)), mimicking genetic alterations of patients suffering from severe forms of Charcot-Marie-Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1(-/-) mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1(-/-) mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1(-/-) mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1(-/-) mice compared with controls. Our findings demonstrate that Charcot-Marie-Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

Show MeSH
Related in: MedlinePlus