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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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Aged Gdap1−/− mice develop a hypomyelinating peripheral neuropathy. (A) Nerve conduction velocity (NCV) measurements of 19-month-old mice with different genotypes are shown as representative original recordings. The broken line represents onset (distal latency) of compound muscle action potentials in unaffected mice. Arrows denote a delayed compound muscle action potential onset (prolonged distal latency) in affected mice. Note that there is no relevant difference in shape and duration of compound muscle action potentials. F-waves are identified by arrowheads. (B) Nerve conduction velocities are represented as means and standard error, n = 6 to 11 animals per genotype and age group; variations of age are indicated as standard error; controls are a group of wild-type and cre-negative animals. n.s. = not significant, *P < 0.05. (C) Transverse ultrathin sections of plantar nerves of 19-month-old Gdap1−/− and P0-cre Gdap1flox/flox (f/f) animals reveal hypomyelination compared to age-matched controls. Scale bars = 5 µm, squares are shown at higher magnification. (D) Quantitative morphological analysis of ultrathin plantar nerve sections at 19 months showing hypomyelination in Gdap1−/− and P0-cre Gdap1flox/flox animals compared with wild-type and cre-negative controls. (E) Quantitative analysis of the total number of myelinated axons in the plantar nerve. (F) The m-ratio, defined as the percentage of the area covered by intra-axonal mitochondria divided by the total area of the axon, is significantly increased only in Gdap1−/− animals. (D and F) One hundred randomly selected myelinated axons were measured per animal. (D–F) represent the means and standard error of independent experiments; cre-negative controls and P0-cre Gdap1flox/flox: n = 3 animals each; wild-type and Gdap1−/−n = 4 animals each; n.s. = not significant, *P < 0.05.
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awt371-F2: Aged Gdap1−/− mice develop a hypomyelinating peripheral neuropathy. (A) Nerve conduction velocity (NCV) measurements of 19-month-old mice with different genotypes are shown as representative original recordings. The broken line represents onset (distal latency) of compound muscle action potentials in unaffected mice. Arrows denote a delayed compound muscle action potential onset (prolonged distal latency) in affected mice. Note that there is no relevant difference in shape and duration of compound muscle action potentials. F-waves are identified by arrowheads. (B) Nerve conduction velocities are represented as means and standard error, n = 6 to 11 animals per genotype and age group; variations of age are indicated as standard error; controls are a group of wild-type and cre-negative animals. n.s. = not significant, *P < 0.05. (C) Transverse ultrathin sections of plantar nerves of 19-month-old Gdap1−/− and P0-cre Gdap1flox/flox (f/f) animals reveal hypomyelination compared to age-matched controls. Scale bars = 5 µm, squares are shown at higher magnification. (D) Quantitative morphological analysis of ultrathin plantar nerve sections at 19 months showing hypomyelination in Gdap1−/− and P0-cre Gdap1flox/flox animals compared with wild-type and cre-negative controls. (E) Quantitative analysis of the total number of myelinated axons in the plantar nerve. (F) The m-ratio, defined as the percentage of the area covered by intra-axonal mitochondria divided by the total area of the axon, is significantly increased only in Gdap1−/− animals. (D and F) One hundred randomly selected myelinated axons were measured per animal. (D–F) represent the means and standard error of independent experiments; cre-negative controls and P0-cre Gdap1flox/flox: n = 3 animals each; wild-type and Gdap1−/−n = 4 animals each; n.s. = not significant, *P < 0.05.

Mentions: In line with our previous results, we found no changes in nerve conduction velocities or compound muscle action potential amplitudes by electrophysiological measurements in 14-month-old Gdap1−/− mice as compared with controls (Fig. 2B and Supplementary Table 1). In contrast, in 19-month-old Gdap1−/− animals, nerve conduction velocities were significantly reduced by 25% compared with age-matched controls (Fig. 2A and B), consistent with a late-onset demyelinating phenotype. After stimulation at the sciatic notch (proximal stimulation), the compound muscle action potential amplitudes were significantly reduced in 19-month-old Gdap1−/− mice when compared with controls, whereas significance was not reached using more distal stimulation at the tibial nerve (Supplementary Table 1). As the compound muscle action potentials were not clearly dispersed, the reduction in the compound muscle action potential amplitudes does not obviously attribute to a pure myelin disorder, but might also reflect an additional axonal phenotype. To investigate axonal pathology of 19-month-old animals, we carried out histological analysis of the plantar nerve, a distal peripheral nerve, as in Charcot–Marie–Tooth disease the longest nerves are the first and most severely affected. Only minor differences appeared in electron micrographs of knockout animals compared with age-matched controls (Fig. 2C). However, morphometric analyses revealed hypomyelination in 19-month-old Gdap1−/− mice compared with controls (Fig. 2D and Supplementary Fig. 2A) with no detectable axonal loss (Fig. 2E). Both, Schwann cells and neurons express GDAP1 in the myelinated peripheral nerves, and it has been proposed that GDAP1 is even mainly expressed by neurons (Niemann et al., 2005; Pedrola et al., 2005, 2008). Thus, to confirm the unexpected predominant myelin disorder phenotype, we disrupted Gdap1 by cre-mediated ablation specifically in Schwann cells (P0-cre Gdap1flox/flox) or in motor neurons (Hb9-cre Gdap1flox/flox). Ablation of GDAP1 in Schwann cells reduced nerve conduction velocities comparable to total knockout animals, whereas motor neuron-specific loss of GDAP1 caused no significant changes when compared with controls (Fig. 2A and B). Moreover, compound muscle action potentials were not decreased in Hb9-cre Gdap1flox/flox animals (3.78 ± 0.41 mV ms; n = 8; controls: 3.0 ± 0.35 mV ms n = 8), indicating that the compound muscle action potential reduction observed in Gdap1−/− mice was likely not due to a motor neuron/axon-autonomous pathology. To further support that ablation of GDAP1 expression in Schwann cells recapitulates the phenotype of Gdap1−/− mice, we analysed also the plantar nerve of 19-month-old P0-cre Gdap1flox/flox mice morphologically. We found significant hypomyelination and no axonal loss in 19-month-old P0-cre Gdap1flox/flox mice comparable to Gdap1−/− mice (Fig. 2D and E). Taken together, our results indicate that loss of GDAP1 expression in Schwann cells is sufficient to cause a hypomyelinating peripheral neuropathy phenotype in aged mice.Figure 2


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)

Aged Gdap1−/− mice develop a hypomyelinating peripheral neuropathy. (A) Nerve conduction velocity (NCV) measurements of 19-month-old mice with different genotypes are shown as representative original recordings. The broken line represents onset (distal latency) of compound muscle action potentials in unaffected mice. Arrows denote a delayed compound muscle action potential onset (prolonged distal latency) in affected mice. Note that there is no relevant difference in shape and duration of compound muscle action potentials. F-waves are identified by arrowheads. (B) Nerve conduction velocities are represented as means and standard error, n = 6 to 11 animals per genotype and age group; variations of age are indicated as standard error; controls are a group of wild-type and cre-negative animals. n.s. = not significant, *P < 0.05. (C) Transverse ultrathin sections of plantar nerves of 19-month-old Gdap1−/− and P0-cre Gdap1flox/flox (f/f) animals reveal hypomyelination compared to age-matched controls. Scale bars = 5 µm, squares are shown at higher magnification. (D) Quantitative morphological analysis of ultrathin plantar nerve sections at 19 months showing hypomyelination in Gdap1−/− and P0-cre Gdap1flox/flox animals compared with wild-type and cre-negative controls. (E) Quantitative analysis of the total number of myelinated axons in the plantar nerve. (F) The m-ratio, defined as the percentage of the area covered by intra-axonal mitochondria divided by the total area of the axon, is significantly increased only in Gdap1−/− animals. (D and F) One hundred randomly selected myelinated axons were measured per animal. (D–F) represent the means and standard error of independent experiments; cre-negative controls and P0-cre Gdap1flox/flox: n = 3 animals each; wild-type and Gdap1−/−n = 4 animals each; n.s. = not significant, *P < 0.05.
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Show All Figures
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awt371-F2: Aged Gdap1−/− mice develop a hypomyelinating peripheral neuropathy. (A) Nerve conduction velocity (NCV) measurements of 19-month-old mice with different genotypes are shown as representative original recordings. The broken line represents onset (distal latency) of compound muscle action potentials in unaffected mice. Arrows denote a delayed compound muscle action potential onset (prolonged distal latency) in affected mice. Note that there is no relevant difference in shape and duration of compound muscle action potentials. F-waves are identified by arrowheads. (B) Nerve conduction velocities are represented as means and standard error, n = 6 to 11 animals per genotype and age group; variations of age are indicated as standard error; controls are a group of wild-type and cre-negative animals. n.s. = not significant, *P < 0.05. (C) Transverse ultrathin sections of plantar nerves of 19-month-old Gdap1−/− and P0-cre Gdap1flox/flox (f/f) animals reveal hypomyelination compared to age-matched controls. Scale bars = 5 µm, squares are shown at higher magnification. (D) Quantitative morphological analysis of ultrathin plantar nerve sections at 19 months showing hypomyelination in Gdap1−/− and P0-cre Gdap1flox/flox animals compared with wild-type and cre-negative controls. (E) Quantitative analysis of the total number of myelinated axons in the plantar nerve. (F) The m-ratio, defined as the percentage of the area covered by intra-axonal mitochondria divided by the total area of the axon, is significantly increased only in Gdap1−/− animals. (D and F) One hundred randomly selected myelinated axons were measured per animal. (D–F) represent the means and standard error of independent experiments; cre-negative controls and P0-cre Gdap1flox/flox: n = 3 animals each; wild-type and Gdap1−/−n = 4 animals each; n.s. = not significant, *P < 0.05.
Mentions: In line with our previous results, we found no changes in nerve conduction velocities or compound muscle action potential amplitudes by electrophysiological measurements in 14-month-old Gdap1−/− mice as compared with controls (Fig. 2B and Supplementary Table 1). In contrast, in 19-month-old Gdap1−/− animals, nerve conduction velocities were significantly reduced by 25% compared with age-matched controls (Fig. 2A and B), consistent with a late-onset demyelinating phenotype. After stimulation at the sciatic notch (proximal stimulation), the compound muscle action potential amplitudes were significantly reduced in 19-month-old Gdap1−/− mice when compared with controls, whereas significance was not reached using more distal stimulation at the tibial nerve (Supplementary Table 1). As the compound muscle action potentials were not clearly dispersed, the reduction in the compound muscle action potential amplitudes does not obviously attribute to a pure myelin disorder, but might also reflect an additional axonal phenotype. To investigate axonal pathology of 19-month-old animals, we carried out histological analysis of the plantar nerve, a distal peripheral nerve, as in Charcot–Marie–Tooth disease the longest nerves are the first and most severely affected. Only minor differences appeared in electron micrographs of knockout animals compared with age-matched controls (Fig. 2C). However, morphometric analyses revealed hypomyelination in 19-month-old Gdap1−/− mice compared with controls (Fig. 2D and Supplementary Fig. 2A) with no detectable axonal loss (Fig. 2E). Both, Schwann cells and neurons express GDAP1 in the myelinated peripheral nerves, and it has been proposed that GDAP1 is even mainly expressed by neurons (Niemann et al., 2005; Pedrola et al., 2005, 2008). Thus, to confirm the unexpected predominant myelin disorder phenotype, we disrupted Gdap1 by cre-mediated ablation specifically in Schwann cells (P0-cre Gdap1flox/flox) or in motor neurons (Hb9-cre Gdap1flox/flox). Ablation of GDAP1 in Schwann cells reduced nerve conduction velocities comparable to total knockout animals, whereas motor neuron-specific loss of GDAP1 caused no significant changes when compared with controls (Fig. 2A and B). Moreover, compound muscle action potentials were not decreased in Hb9-cre Gdap1flox/flox animals (3.78 ± 0.41 mV ms; n = 8; controls: 3.0 ± 0.35 mV ms n = 8), indicating that the compound muscle action potential reduction observed in Gdap1−/− mice was likely not due to a motor neuron/axon-autonomous pathology. To further support that ablation of GDAP1 expression in Schwann cells recapitulates the phenotype of Gdap1−/− mice, we analysed also the plantar nerve of 19-month-old P0-cre Gdap1flox/flox mice morphologically. We found significant hypomyelination and no axonal loss in 19-month-old P0-cre Gdap1flox/flox mice comparable to Gdap1−/− mice (Fig. 2D and E). Taken together, our results indicate that loss of GDAP1 expression in Schwann cells is sufficient to cause a hypomyelinating peripheral neuropathy phenotype in aged mice.Figure 2

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