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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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Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. (A) Spinal cords of 19-month-old wild-type or Gdap1−/− animals were fractionated by differential centrifugation. In homogenates from Gdap1−/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. (B) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, **P < 0.005. (C) The domain structure of GDAP1L1 is illustrated with focus on the carboxy-terminal domains harbouring the hydrophobic domain 1 (HD1) and the transmembrane domain (TMD; GST-N and GST-C: domains conserved in glutathione S-transferases). The tail-anchored domain is formed by the transmembrane domain and surrounding positively charged amino acids. (D) N1E-115 cells were infected with lentiviral particles encoding non-silencing short-hairpin RNA (shRNA control) or short-hairpin RNA against Gdap1 (shGDAP1) or were left uninfected. When the knockdown was detectable (Supplementary Fig. 7A and B), cells were transiently co-transfected with expression constructs encoding mitochondrially targeted DsRed together with mitochondrially targeted green fluorescent protein (mtGFP) as negative control, with GDAP1 as positive control, with GDAP1L1 or with GDAP1L1 340X, lacking the potential tail-anchor domain of GDAP1L1. The next day cells were fixed and stained. In blinded countings, the mitochondrial morphology was assessed by mitochondrially targeted DsRed fluorescence (Niemann et al., 2005). One hundred randomly selected cells were analysed per experiment and condition, means and standard error of the mean, n = 3, n.s. = not significant, **P < 0.01.
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awt371-F7: Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. (A) Spinal cords of 19-month-old wild-type or Gdap1−/− animals were fractionated by differential centrifugation. In homogenates from Gdap1−/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. (B) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, **P < 0.005. (C) The domain structure of GDAP1L1 is illustrated with focus on the carboxy-terminal domains harbouring the hydrophobic domain 1 (HD1) and the transmembrane domain (TMD; GST-N and GST-C: domains conserved in glutathione S-transferases). The tail-anchored domain is formed by the transmembrane domain and surrounding positively charged amino acids. (D) N1E-115 cells were infected with lentiviral particles encoding non-silencing short-hairpin RNA (shRNA control) or short-hairpin RNA against Gdap1 (shGDAP1) or were left uninfected. When the knockdown was detectable (Supplementary Fig. 7A and B), cells were transiently co-transfected with expression constructs encoding mitochondrially targeted DsRed together with mitochondrially targeted green fluorescent protein (mtGFP) as negative control, with GDAP1 as positive control, with GDAP1L1 or with GDAP1L1 340X, lacking the potential tail-anchor domain of GDAP1L1. The next day cells were fixed and stained. In blinded countings, the mitochondrial morphology was assessed by mitochondrially targeted DsRed fluorescence (Niemann et al., 2005). One hundred randomly selected cells were analysed per experiment and condition, means and standard error of the mean, n = 3, n.s. = not significant, **P < 0.01.

Mentions: Long-term loss of GDAP1 expression causes mild oxidative stress conditions in peripheral nerves (Fig. 4A and B) and in cultured neuronal cell lines (Noack et al., 2012). Therefore, we carried out differential centrifugation to enrich mitochondria from the spinal cord to obtain evidence whether translocation of GDAP1L1 occurs also in vivo. In the spinal cord of 19-month-old Gdap1−/− mice, equal amounts of GDAP1L1 were expressed compared with age-matched controls (Fig. 7A and B). However, significantly more GDAP1L1 co-sedimented with Porin compared with the controls (Fig. 7A and B).Figure 7


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)

Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. (A) Spinal cords of 19-month-old wild-type or Gdap1−/− animals were fractionated by differential centrifugation. In homogenates from Gdap1−/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. (B) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, **P < 0.005. (C) The domain structure of GDAP1L1 is illustrated with focus on the carboxy-terminal domains harbouring the hydrophobic domain 1 (HD1) and the transmembrane domain (TMD; GST-N and GST-C: domains conserved in glutathione S-transferases). The tail-anchored domain is formed by the transmembrane domain and surrounding positively charged amino acids. (D) N1E-115 cells were infected with lentiviral particles encoding non-silencing short-hairpin RNA (shRNA control) or short-hairpin RNA against Gdap1 (shGDAP1) or were left uninfected. When the knockdown was detectable (Supplementary Fig. 7A and B), cells were transiently co-transfected with expression constructs encoding mitochondrially targeted DsRed together with mitochondrially targeted green fluorescent protein (mtGFP) as negative control, with GDAP1 as positive control, with GDAP1L1 or with GDAP1L1 340X, lacking the potential tail-anchor domain of GDAP1L1. The next day cells were fixed and stained. In blinded countings, the mitochondrial morphology was assessed by mitochondrially targeted DsRed fluorescence (Niemann et al., 2005). One hundred randomly selected cells were analysed per experiment and condition, means and standard error of the mean, n = 3, n.s. = not significant, **P < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927703&req=5

awt371-F7: Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. (A) Spinal cords of 19-month-old wild-type or Gdap1−/− animals were fractionated by differential centrifugation. In homogenates from Gdap1−/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. (B) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, **P < 0.005. (C) The domain structure of GDAP1L1 is illustrated with focus on the carboxy-terminal domains harbouring the hydrophobic domain 1 (HD1) and the transmembrane domain (TMD; GST-N and GST-C: domains conserved in glutathione S-transferases). The tail-anchored domain is formed by the transmembrane domain and surrounding positively charged amino acids. (D) N1E-115 cells were infected with lentiviral particles encoding non-silencing short-hairpin RNA (shRNA control) or short-hairpin RNA against Gdap1 (shGDAP1) or were left uninfected. When the knockdown was detectable (Supplementary Fig. 7A and B), cells were transiently co-transfected with expression constructs encoding mitochondrially targeted DsRed together with mitochondrially targeted green fluorescent protein (mtGFP) as negative control, with GDAP1 as positive control, with GDAP1L1 or with GDAP1L1 340X, lacking the potential tail-anchor domain of GDAP1L1. The next day cells were fixed and stained. In blinded countings, the mitochondrial morphology was assessed by mitochondrially targeted DsRed fluorescence (Niemann et al., 2005). One hundred randomly selected cells were analysed per experiment and condition, means and standard error of the mean, n = 3, n.s. = not significant, **P < 0.01.
Mentions: Long-term loss of GDAP1 expression causes mild oxidative stress conditions in peripheral nerves (Fig. 4A and B) and in cultured neuronal cell lines (Noack et al., 2012). Therefore, we carried out differential centrifugation to enrich mitochondria from the spinal cord to obtain evidence whether translocation of GDAP1L1 occurs also in vivo. In the spinal cord of 19-month-old Gdap1−/− mice, equal amounts of GDAP1L1 were expressed compared with age-matched controls (Fig. 7A and B). However, significantly more GDAP1L1 co-sedimented with Porin compared with the controls (Fig. 7A and B).Figure 7

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