2 Department of Neuroscience, Ophthalmology and Genetics, and Center of Excellence for Biomedical Research, University of Genoa, 16132 Genoa, Italy
Correspondence to Ueli Suter: usuter{at}cell.biol.ethz.ch
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Introduction |
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The available information about the biology of GDAP1 is scarce. GDAP1 was identified as a transcript that was up-regulated after ganglioside-induced cholinergic differentiation of the mouse neuroblastoma cell line Neuro2a (Liu et al., 1999). Furthermore, an increase of GDAP1 mRNA expression also was found in neural-differentiated P19 cells and during development of the mouse brain (Liu et al., 1999).
Some hints about the molecular function of GDAP1 have been provided by bioinformatic analyses (Marco et al., 2004) confirming and extending previous reports (Baxter et al., 2002; Cuesta et al., 2002; Nelis et al., 2002). These studies demonstrated that GDAP1 and related proteins in invertebrates and vertebrates contain the characteristic domains of GSTs (GST-N, GST-C). Based on its particular domain features, GDAP1 was proposed as the founder of a new GST family. Members of this family are characterized by an enlarged interdomain between the GST-N and GST-C domains, and contain COOH-terminal hydrophobic stretches with potential transmembrane features (Marco et al., 2004).
Here we show that Schwann cells and neurons of myelinated peripheral nerves express GDAP1. Thus, both cell types might be affected directly by the consequences of the disease-causing mutations in GDAP1. Within the cell, GDAP1 functions as a tail-anchored protein of the mitochondrial outer membrane and promotes fragmentation of mitochondria. On this basis, we define GDAP1 as a novel regulator of the mitochondrial network. This ability is lost or reduced in CMT-associated mutated forms of GDAP1 and is very likely to contribute to the axonal and/or demyelinating defects that are seen in patients. The precise regulation of fusion and fission of mitochondria seems to be crucial for the integrity of the peripheral nervous system (PNS).
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Results |
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GDAP1 is a mitochondrial protein
We next determined the precise subcellular localization of GDAP1. In COS-7 cells that were transfected with an untagged GDAP1 full-length expression construct, we found GDAP1 in distinct structures with tubular and vesicular appearances throughout the cytoplasm at the onset of expression (Fig. 2 A). These GDAP1-positive formations were identified as mitochondria by congruent costaining with the mitochondrial markers MitoTracker (Fig. 2 A, ad), cytochrome c (Fig. 4 B), and porin (not depicted). No appreciable overlap was found with other membranous compartments, including the Golgi complex (marker giantin; Fig. 2 A, eh) or the ER (marker protein disulfide isomerase [PDI]; Fig. 2 A, il).
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To support further the localization of GDAP1 to mitochondria, we used a cell fractionation approach. Mitochondria were enriched by differential centrifugation (Graham, 2004), and the various steps were analyzed by Western blotting. Cell fractionations were performed with transfected Cos-7 cells (not depicted) and with endogenously GDAP1-expressing N1E-115 cells (Fig. 2 B). As expected, GDAP1 levels were enhanced specifically by this procedure in parallel with porin, an abundant transmembrane protein of the outer mitochondrial membrane, and in contrast to proteins of the ER, like PDI. Thus, GDAP1 is located in mitochondria.
GDAP1 is an integral membrane protein of the outer mitochondrial membrane
To elucidate the localization of GDAP1 within the mitochondria, we purified mitochondria from COS-7 cells, 24 h after transfection with a GDAP1 expression construct. Mitochondria were resuspended and digested with an excess of proteinase K in the presence or absence of detergent (Olichon et al., 2002), and analyzed by Western blot. The GDAP1 signal was lost upon proteinase incubation, whereas cytochrome c, located in the intermembrane space, was protected and disappeared only if increasing amounts of digitonin or SDS were added (Fig. 3 A). Because the antisera used in this analysis recognize the NH2-terminal proximal segment of GDAP1 (Fig. S1), these data indicate that the NH2 terminus of GDAP1, and thereby, the GST domains, are exposed to the cellular cytoplasm. In support of this interpretation, we found GDAP1 in the outer mitochondrial membrane by ultrastructural analysis using immunogold labeling (unpublished data).
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GDAP1 regulates the architecture of mitochondria
In various experiments, we observed time-dependent changes in the mitochondrial morphology upon GDAP1 transfection. With prolonged expression of GDAP1, the mitochondria in COS-7 cells became more fragmented, and tubular forms were less prominent (Fig. 4 A, c and d). This effect was not seen in mock-transfected cells (Fig. 4 A, a). We reasoned that overexpression of any protein of the outer mitochondrial membrane could cause this effect. However, mitochondrial morphology was not appreciably altered upon transient expression of the GFP-tagged human transporter of the outer membrane subunit 7 (EGFP-hTOM7), in agreement with previous results (Stojanovski et al., 2004) (Fig. 4 A, b). This indicates a specific effect of GDAP1 in our experimental settings. Thus, we quantified the morphologic alterations caused by GDAP1 overexpression. To this end, we defined five distinct mitochondrial architectures within cells (for examples, see Fig. 4 A, e, bottom panel): perinuclear aggregated ("aggregated"), predominantly tubular ("tubular"), tubular and vesicular ("mixed"), predominantly vesicular but with some tubular structures ("vesicular"), and completely vesicular or fragmented mitochondria ("fragmented"). This analysis revealed that 15 h after transfection with GDAP1, fragmented mitochondria increase significantly at the expense of tubular and mixed mitochondrial formations compared with control cells (Fig. 4 A, e). As suspected from our previous impressions, this shift toward mitochondrial fragmentation was even more pronounced 20 h after transfection.
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Loss of the mitochondrial transmembrane potential (m) also triggers the fragmentation of mitochondria (Legros et al., 2002). Thus, we asked whether the fragmentation that is induced by overexpression of GDAP1 might be secondary to an effect on
m. The uptake of the dye, MitoTrackerH2XRos, into mitochondria is dependent on mitochondrial activity (Chen and Cushion, 1994), which provides a mean to test this possibility (Fig. 4 C, a and b). Transient transfection with low amounts of an EGFP-hFis1 expression construct served as positive control (Yoon, 2004). Dye uptake was unchanged in GDAP1-expressing cells compared with nontransfected HeLa cells, whereas it was reduced to 70% in EGFP-hFis1positive HeLa cells. In addition, we generated stably transfected Neuro2a cells that inducibly overexpress GDAP1. Using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide in flow cytometry (Cossarizza et al., 1993), we confirmed that overexpression of GDAP1 does not induce apoptosis or influence
m in these cells (unpublished data).
GDAP1-induced mitochondrial fragmentation is blocked by dynamin-related protein 1 (K38A) coexpression and counterbalanced by mitofusin 1 and 2
The shape of mitochondria depends on the balance of fusion and fission. Thus, we tested whether the induction of fragmentation by GDAP1 can be counterbalanced by proteins that are known to induce fusion or block fission. The dynamin-related protein 1 (Drp1) plays a central role in the fission of mitochondria; the dominant-negative Drp1(K38A) form blocks mitochondrial fission (Smirnova et al., 1998). We coexpressed GDAP1 with Drp1 or Drp1(K38A) in COS-7 cells, and analyzed the mitochondrial morphology of cells that express both proteins 17 h after transfection. Western blotting confirmed comparable expression levels of the transfected constructs (unpublished data). Coexpression of GDAP1 and Drp1 did not influence GDAP1-induced mitochondrial fragmentation considerably (Fig. 5 A, af). This outcome was expected, because overexpression of Drp1 per se does not change the mitochondrial architecture; there is an abundant pool of endogenous Drp1 in the cell (Yoon, 2004). In contrast, if coexpressed with GDAP1, Drp1(K38A) partially inhibited the fragmentation of mitochondria (Fig. 5 A, gi). An increased number of GDAP1-Drp1(K38A) doubly expressing cells still had fragmented mitochondria compared with EGFP-hTOM7 control-transfected cells; however, this number was reduced significantly compared with GDAP1-only expressing cells (Fig. 5 A, j). Also, more cells than controls showed extended tubular mitochondrial morphology, the most prominent phenotype observed when Drp1(K38A) is expressed alone (85%; unpublished data). In summary, coexpression of Drp1(K38A) can block GDAP1-induced fragmentation partially, but without restoring the normal pattern completely.
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GDAP1 does not reduce mitochondrial fusion
Our previous experiments have shown that the overexpression of GDAP1 promotes mitochondrial fragmentation. This mitochondrial morphology might result from increased mitochondrial fission, reduced mitochondrial fusion, or a combination of both effects. Using polyethylene glycol (PEG)-based cell fusion technique, we examined the effect of GDAP1 on mitochondrial fusion (Mattenberger et al., 2003). HeLa cells that were transfected transiently with mitochondrial targeted DsRed (mtDsRed) were coplated with HeLa cells that were transfected transiently with mitochondrial targeted GFP (mtGFP; see Fig. 7, ac) or GDAP1 (see Fig. 7, df). The cells were fused with PEG and cultivated in the presence of cycloheximide. As reported previously, we found mitochondrial fusion in hybrids of cells transfected with mtDsRed fused to cells transfected with mtGFP 34 h after cell fusion. This process was almost complete 6 h after cell fusion (Fig. 6, ac, g; Mattenberger et al., 2003). An identical timing and extent of mitochondrial fusion was observed in cell hybrids of cells that were transfected with mtDsRed fused with cells transfected with GDAP1 (Fig. 6, dg). Thus, GDAP1 overexpression does not interfere significantly with mitochondrial fusion, and indicates that GDAP1 is a fission-inducing factor.
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Disease-associated point mutations affect the ability of GDAP1 to induce fragmentation of mitochondria
Mutations leading to motor and sensory neuropathies are likely to yield important information about particular GDAP1 domains that are required for the proper function of the protein. Thus, we tested various GDAP1 point mutations (Fig. 9 A; M116H, R120Q, R161H, R282C, R310Q), in addition to the truncation mutants that were described above. Each of the selected point mutant forms of GDAP1 protein was targeted to mitochondria in transfected COS-7 cells (for examples, see Fig. 9 B). However, most interestingly, the influence on the mitochondrial architecture differed among the point mutants tested. Each mutant showed some impairment in the ability to induce fragmentation compared with wild-type GDAP1 protein, as indicated by the variable reduction of the fraction of cells with only fragmented mitochondria (Fig. 9 C, a). The mutants R161H and R310Q were completely inactive with respect to the promotion of mitochondrial fragmentation, and showed morphologies that were comparable to those seen in control transfections. We conclude that GDAP1 mutations that are found in patients who have CMT are defective in the ability of GDAP1 to induce mitochondrial fragmentation, albeit to different degrees.
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Discussion |
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It is well-established that the nervous system is especially sensitive to mitochondrial dysfunction (Bouillot et al., 2002; Bossy-Wetzel et al., 2003; Finsterer, 2004). Furthermore, a critical role for mitochondria is discussed in the context of diseases and syndromes with liability to neuropathies, including diabetes (Vincent et al., 2004) or degeneration related to ageing (Bossy-Wetzel et al., 2003). We have found that GDAP1, the product of the disease-causing gene in particular forms of the neurodegenerative disorder CMT, is a key regulator of mitochondrial fragmentation. The observed fragmentation effect most likely is linked closely to mitochondrial fission. This important specific aspect of the biology of mitochondria has been appreciated fully only recently (Mozdy and Shaw, 2003; Chen and Chan, 2004; Rube and van der Bliek, 2004). In relation to diseases, two proteins that are involved in mitochondrial fusion are involved in neuropathies (Bossy-Wetzel et al., 2003). Mutations in the dynamin-related GTPase, OPA-1, lead to optic atrophy (Delettre et al., 2000); mutations in MFN2 were linked recently to CMT2A (Zuchner et al., 2004). We describe that GDAP1 also takes part in the regulation of mitochondrial dynamics by promoting mitochondrial fission. The fission effect is not secondary as a result of the induction of apoptosis or the reduction of the m. GDAP1-induced mitochondrial fragmentation was counteracted by the fusion inducing factors, Mfn1 and Mfn2 and the dominant negative fission-blocking Drp1(K38A). PEG-mediated cell fusions further demonstrated that GDAP1 expression does not interfere with mitochondrial fusion. These findings suggest that common elements in the disease mechanisms of CMT are caused by mutations in GDAP1 and MFN2, although GDAP1 and Mfn2 have opposite effects on the mitochondrial morphology. The relation between GDAP1 and Mfn2 is intriguing if the two phenotypes of the associated diseases are compared. Mfn2 has been described exclusively in the context of axonal neuropathies in CMT2A, which indicates a neuronal basis of the disease mechanism. GDAP1 has been associated with axonal, demyelinating, and mixed phenotypes in CMT. Thus, a neuronal disease origin also might be favored in CMT caused by mutations in GDAP1 if an altered mitochondrial regulation by mutations in GDAP1 and MFN2 is put forward as the hypothetic basis of both genetically distinct disorders. However, we found that GDAP1 is expressed by myelinating Schwann cells as well as motor and sensory neurons; it is possible that the disease primarily affects Schwann cells, neurons, or both. Cell-type specific elimination of GDAP1 individually from neurons or Schwann cells will be required to resolve this issue.
Truncations and point mutations in GDAP1 are associated with CMT. Our results indicate that the COOH-terminal truncations that were tested have lost mitochondrial localization, whereas the examined GDAP1 proteins carrying missense mutations remained correctly targeted to mitochondria. These data are consistent with a recent report on the subcellular localization and effects of some GDAP1 mutations (Pedrola et al., 2005). We further demonstrate that GDAP1 proteins carrying disease-related mutations have lost the ability to induce mitochondrial fragmentation to various degrees. However, we have been unable to correlate the activity differences to disease onset, severity, and/or pathology. The variability of the phenotypes seems to be too high, even between relatives with the identical mutation (Azzedine et al., 2003).
The mitochondrial network is dynamic, and shifting the balance of fusion and fission is a major regulatory mechanism. Although the functional consequences of these events are understood poorly, it is obvious that complex and divergent stimuli influence the mitochondrial appearance (Goglia et al., 1999; Bach et al., 2003; Bossy-Wetzel et al., 2003; Honda and Hirose, 2003). The finding that mutations in genes affect this process in hereditary motor and sensory neuropathies suggests that myelinated peripheral nerves are particularly dependent on the proper function and control of this system. Whether this vulnerability is the consequence of a deregulation of the mitochondrial architecture or an impaired mitochondrial transport (Shy, 2004) remains to be determined.
Recently, evidence for mitochondrial fusion intermediates and an Mfn complex mediating mitochondrial tethering was provided (Koshiba et al., 2004; Meeusen et al., 2004). How the potential GST protein GDAP1, might, on a molecular level, be involved in the regulation of the mitochondrial architecture remains elusive. On a highly speculative note, the mitochondrial morphology is dependent on glutathione (GSH). Depletion of GSH leads to mitochondrial fusion, and results in a tubular mitochondrial network (Soltys and Gupta, 1994), although this effect may not be based solely on the GSH concentration (Bowes and Gupta, 2005). It remains unknown whether these are genuine hints that GDAP1 is a mitochondrial fission-inducing factor that is dependent on GSH.
In summary, we introduce GDAP1 as a key regulator of mitochondrial dynamics. If this activity is lost, the integrity of peripheral nerves is disturbed and leads to myelin and axonal defects. We anticipate that further elucidation of the cellular and molecular mechanisms that underlie the pathology of GDAP1-based CMT will help to uncover the function of GDAP1 and mitochondrial dynamics in peripheral nerves. Conversely, further examination of the molecular function of GDAP1 in the biology of mitochondria will contribute significantly to our basic understanding of CMT.
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Materials and methods |
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Constructs
GDAP1 cDNA was amplified by RT-PCR from adult mouse brain using the following primers: 5'-CGGATCCATGGCTCGGAGGCAGGAC-3', 5'-CGTCGACTAGAAATAATTTGGTCTGG-3'. GDAP1-like-1 cDNA was cloned using 5'-CGGATCCATGGCGACCCCCAAC-3' and 5'-CCCAGCCAGCTCTAGATGT-3'. Point mutations and truncations were generated by PCR with appropriate primer pairs. All cDNAs were cloned into the pGEM-T vector and verified by sequencing. The cDNAs were subcloned into pcDNA3.1 (Invitrogen) or EGFP-C1 (CLONTECH Laboratories, Inc.). Alternatively, the coding region for EGFP was replaced by the HA epitope (HA-C1 vector, provided by A. Hirschy, Swiss Federal Institute of Technology, Zürich, Switzerland). EGFP-hTOM7 and EGFP-hFis1 expression constructs were provided M. Ryan (La Trobe University, Melbourne, Australia). Mfn1-10xMyc, Mfn1(K88T)-10xMyc, Mfn2-16xMyc, Mfn2(K109A)-16xMyc, Drp1-6xMyc, and Drp(K38A)6xMyc were provided by D. Chan (California Institute of Technology, Pasadena, California). MtDsRed2 and mtGFP were from CLONTECH Laboratories, Inc.
Cell culture
Rat Schwann cells were prepared essentially as described by Brockes et al. (1979). Cells were grown in Schwann cell medium (DME; Invitrogen), containing 10% FCS, 4 µg/ml glial growth factor (crude pituitary extract (Sigma-Aldrich), and 4 µM forskolin (Sigma-Aldrich). For growth arrest, cells were incubated for 3 d in low serum medium (DME/F12 medium (Invitrogen), 0.5% FCS, 4 µM forskolin, 100 µg/ml human apo-transferrin, 60 ng/ml progesterone, 16 µg/ml putrescine, 10 µg/ml insulin, 400 ng/ml L-thyroxin, 160 ng/ml selenium, 0.02 ng/ml triiodothyronine, and 300 µg/ml BSA (Fluka). Supplements were from Sigma-Aldrich, unless stated otherwise. COS-7 cells, HeLa cells, and N1E-115 cells were maintained in DME containing 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). COS-7 and HeLa cells were transfected using Fugene 6, according to the recommendations of the manufacturer (Roche). RNAi transfections on N1E-115 cells were done with 1.7 µg/ml lipofectamine 2000 (Invitrogen) and 55 nM Stealth RNAi (Invitrogen; GDAP1-specific: GGCCA CUCAG AUCAU UGAUU AUCUU; control: GGCAC UCCUA GGUUA AUUAU ACCUU). The cells were fixed or harvested 48 h after the start of transfection.
Cell fractionation
The differential centrifugation protocol to enrich for mitochondria was adopted from Graham (2004). Proteinase digestion was performed as described elsewhere (Olichon et al., 2002). Proteinase K (50 µg/ml) was added to the cell fractionation buffer with increasing concentrations of digitonin, or SDS. After 30 min on ice, the digest was stopped with 0.2 mM PMSF. To test for membrane integration of GDAP1, the first mitochondrial pellet from the differential centrifugation procedure was split and resuspended with a vortex mixer for 30 s at full speed in four different buffers: the homogenization buffer with 0.25 M sucrose (control), the control buffer plus 1 M sodium chloride (1 M NaCl), the control buffer plus 0.1% Triton X-100, or in 0.1 M sodium carbonate pH 11.0 (carbonate). The samples were centrifuged again for 20 min at 8000 g, 4°C. Equal volumes of the supernatants and pellets were analyzed by Western blot (Olichon et al., 2002; Stojanovski et al., 2004).
Cell fusions
For cell fusion, HeLa cells were transfected transiently with mtDsRed, mtGFP, or GDAP1. 8 h after the start of transfection, the cells were coplated on coverslips and cultivated overnight. The next morning, cycloheximide was added to the medium (1 mM). After 30 min, the cells were fused on a drop of prewarmed 50% w/v PEG 6000 (Sigma-Aldrich) in PBS/glucose (1 g/l) for 2 min. The cells were washed intensively with PBS/glucose and were kept in cultivation medium plus cycloheximide.
Antibodies
Polyclonal rabbit anti-GDAP1 antisera were generated by Pineda. Two different peptides were used as antigen (peptide 1: MARRQDEARAGVPL; peptide 2: FLDERTPRLMPDEGS). The antisera were used in a 1:7,500 dilution for Western blotting and 1:1,000 for immunofluorescence. Unless indicated otherwise, the sera against peptide 1 (serum 1) and peptide 2 (serum 2) were used and gave identical results. For immunohistochemistry of paraffin sections, we used serum 1, which was purified with the Montage antibody purification Kit (Millipore). The monoclonal mouse antibodies against ß actin (clone AC-74), neurofilament 160 (clone NN18), and S-100 ß-subunit (clone SH-B1) were from Sigma-Aldrich. Monoclonal anti-cytochrome c antibody (clone 7H8.2C12 and clone 6H2.B4) and the polyclonal rabbit anti-caspase3 antibody were obtained from BD Biosciences. The monoclonal rat anti-HA (clone 3F10) was purchased from Roche. The polyclonal rabbit anti-PDI antisera were provided by Dr. A. Helenius (Swiss Federal Institute of Technology, Zürich, Switzerland).
Immunohistochemistry
Cells were fixed with 2.5% PFA/PBS for 30 min at room temperature. Tissue was fixed overnight with 3.7% formalin, dehydrated, and embedded in paraffin in a Tissue Processor TP1010 (Leica). 6-µm sections were prepared using a Rotary Microtome HM 330 (Microm). For immunohistochemistry, fixed cells and rehydrated sections were incubated with 0.1% Triton X-100/PBS for 10 min. After washing with PBS, the specimens were incubated with 10% goat serum in PBS for 1 h at room temperature, followed by incubation with primary antibodies in 10% goat serum/PBS for 90 min at room temperature or overnight at 4°C. After washing, samples were exposed to appropriate secondary antibodies. Cy3- and Cy5-conjugated antibodies were from the Jackson Laboratory, secondary antibodies with Alexa-dyes from Molecular Probes. Samples were incubated with DAPI (Roche) and mounted in Immu-Mount (ThermoShandon). Specimens were analyzed with a Zeiss Axiophot microscope (Carl Zeiss Microimaging, Inc.). Alternatively, we used confocal microscopy on an inverted microscope DM IRB/E equipped with a true confocal scanner TCS SP1, a PL APO 63x/1.32 oil immersion objective (Leica) as well as argon, heliumneon lasers. Image processing was done on a Silicon Graphics workstation using Imaris (Bitplane AG). For labeling of mitochondria, we used MitoTracker Red (Molecular Probes) following the manufacturer's recommendations. MitoTracker Red CM-H2XRos was added to the medium in a final concentration of 0.5 µM for 30 min. After this pulse, the cells were cultivated in growth medium without dye for 10 min before fixation. To determine the relative uptake of MitoTrackerH2XRos into transfected cells, we analyzed single-plane confocal images. Using ImageJ, we measured the relative fluorescence (RF) intensity of MitoTrackerH2XRos and the area. We determined the RF/area in transfected cells and in untransfected cells on the same picture. For each picture, the RF/area of transfected cells was divided by the RF/area of untransfected cells. The average and the standard deviation of several pictures (n 6) were determined using two-tailed unpaired t test.
Western blotting
After SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membrane (Millipore). Blots were blocked with 10% nonfat dry milk powder in TBS. The primary antibodies were diluted according to the manufacturer's recommendation or as described above. Blots were washed with 0.05% Tween 20/TBS. Secondary antibodies were from Santa Cruz Biotechnology, Inc., DakoCytomation, and Southern Biotechnology Associates, Inc. Immunoreactive bands were visualized by Western Lightning (PerkinElmer) or CDP-Star (Roche).
Online supplemental material
Fig. S1 shows validation of GDAP1-specific antisera. Fig. S2 shows that endogenous GDAP1 is found in mitochondria of Schwann cells and PNS neurons.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation and the National Center of Competence in Research "Neural Plasticity and Repair" to U.S. and FIRB-RBAU01KJE4_002 to A. Schenone.
Submitted: 18 July 2005
Accepted: 22 August 2005
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