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Normal Mitochondrial DNA and Respiratory Chain Activity in Familial Dysautonomia Fibroblasts
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
59, 20–27 (1996)
0059
Normal Mitochondrial DNA and Respiratory Chain Activity in Familial Dysautonomia Fibroblasts PAULA STRASBERG,*,†,‡ PHILLIPA BRIDGE,* FRANK MERANTE,§ HERMAN YEGER,Ø
AND
JUDE PEREIRA*
*Division of Neuroscience, The Research Institute, and †Department of Clinical Genetics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; ‡Department of Clinical Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; §Center for Cardiovascular Research, General Division, The Toronto Hospital, Toronto, Ontario, Canada; and Ø Department of Pathology, The Hospital for Sick Children, and The University of Toronto, Toronto, Ontario, Canada Received November 7, 1995, and in revised form March 29, 1996
III), with features of absent lingual fungiform papillae, lack of tearing, vasomotor instability, increased sweating, cold hands and feet, corneal anesthesia, neurological defects, respiratory complications, erythematous blotching of the skin, and scoliosis (1–3), restricted to Ashkenazi Jews. In sural nerve, there is a decrease in the number of myelinated fibers (4). Pathognomic for FD is the fact that FD patients do not exhibit the wheal and flare response to intradermally injected histamine (5). The gene (DYS) maps to chromosome 9q31 (6) (6–10). Previously, we found increases in Gb3 in FD fibroblasts and lymphoblasts and a decrease in ganglioside levels, as well as light microscopic pleiomorphic changes in FD fibroblasts, suggestive of changes in the plasma membrane (7). We described an increase in Gb3 on the surface of synchronized cells at the G1/ S boundary of the cell cycle, based on Gb3 –verotoxin (derived from E. coli) interactions (8). Using D-glucosamine-1-14C as an in vitro precursor, we reported a marked decrease in the rate of incorporation of Dglucosamine into the sialic acid and the N-acetylgalacto/glucosamine moieties of gangliosides and neutral GSL in intact FD compared to control lymphoblasts. The total ganglioside content of FD lymphoblasts (primarily GM3 , measured as incorporation of 3H from NaB3H4) was also decreased. These data indicated differences in the turnover of sialic acid and N-acetylated sugar constituents in FD versus normal cells (9). Earlier experiments in our laboratory demonstrated that cerebrosides and lysocerebrosides, at concentrations approaching those in lysosomal stor-
Familial dysautonomia (FD), an autosomal recessive disease mapped to chromosome 9q31, is a sensory and autonomic neuropathy of unknown etiology. We have previously reported light microscopic pleiomorphic changes in cells suggestive of altered plasma membranes, an increase in globotriaosylceramide (Gb3), reflected by an increase in Gb3 on the surface of the plasma membrane, and a decrease in the rate and amount of ganglioside synthesized. In unrelated studies, we demonstrated that storage of glycospingolipids (GSL) is deleterious to mitochondrial function. Recently, mitochondrial dysfunction has been associated with neurodegenerative disease, superimposed on an autosomal inheritance pattern. We have now probed Southern blots of total FD fibroblast DNA, digested with BamHI, EcoRII, and/or PvuII, with purified placental 32P-labeled mitochondrial DNA. The sizes of all FD mitochondrial DNAs were normal (16,569 bp), some containing previously identified BamHI polymorphisms. Lactate/ pyruvate ratios, and activities of Complexes II and III, matched those of control cells. Electron microscopy revealed morphologically normal mitochondria, in conjunction with a normal oxidative state, determined using the redox dyes Mito Tracker CMXR and CMXR-H2 and fluorescence microscopy. We conclude that mitochondrial dysfunction, due to GSL accumulation, changes in mitochondrial DNA, or mutation of a chromosome 9q gene involved in mitochondrial function, is neither a primary nor a secondary cause of FD, as determined by a study of FD fibroblasts. q 1996 Academic Press, Inc.
Familial dysautonomia (FD, MIM 223900) is a hereditary sensory and autonomic neuropathy (HSAN20 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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age diseases (LSD), inhibited oxidative phorphorylation and caused mitochondrial swelling in a manner suggestive of distortion of the mitochondrial membranes (10–12). Furthermore, it is generally accepted that there is a loss of neurons and myelin in the vicinity of accumulations of storage cells (13). It has more recently been shown that GSL or their modified catabolites modulate transmembrane signal transduction at the cell surface, regulate cell proliferation, induce differentiation, and serve as receptors for cellular interactions, bacterial toxins, and hormones (14). Ceramide has been cited as a potential second messenger in the nervous system (15) and as a stress signal and mediator of growth suppression and apoptosis (16). Recently, mitochondrial dysfunction has been associated with neurodegenerative disease, superimposed on an autosomal inheritance pattern, for example, in Alzheimer and Parkinson diseases, and diabetes mellitus, due to mitochondrial DNA mutagenesis from oxygen radical generation (17,18). In the case of Batten disease, there is accumulation of subunit c of the mitochondrial ATP synthase, a protein derived from nuclear DNA (19). This may not be the primary event in the etiology of Batten disease, but nonetheless represents a form of mitochondrial dysfunction associated with a genetic disease. Our studies previously indicated alterations in GSL in FD with the potentially deleterious effects on mitochondria noted above. Normally glucosamine is incorporated into gangliosides as CMP-sialic acid and high-energy UDP sugars, both of which cannot be synthesized without ATP. Since alterations in mitochondrial function have been a primary cause of (e.g., neonatal lacticacidemia (20), MELAS (21), MERRF (22)), or a secondarily related finding in, other neurological diseases, we decided to investigate mitochondrial function and the integrity of mitochondrial DNA in FD.
21
mal births was isolated and purified using a Ficoll gradient as described elsewhere (24). DNA was extracted from the mitochondria as above. Random priming of mitochondrial DNA. Mitochondrial DNA was linearized by digestion with BamHI, reprecipitated, taken up in TE (10 mM Tris– HCl, 1 mM EDTA, pH 7.4) (23), and labeled with 32P by random priming using the Boehringer Mannheim random primed kit (Model 1004760) according to manufacturer’s instructions. Southern blotting of fibroblast DNA. Two micrograms of DNA per sample was digested with BamHI, PvuII, or BamHI plus EcoRI overnight (in a volume of 200 ml), precipitated, redissolved, and loaded with tracking dye onto 0.6% agarose gels, along with appropriate size standards. Gel electrophoresis and Southern transfer were done according to standard procedures (23). Blots were hybridized with the 32Pmitochondrial DNA (2.5 1 106 cpm per ml, 10 ml per blot), washed with 0.5 1 SSPE (23), 0.1% SDS for 30 min at 427C and 15 min at 657C, and exposed to Kodak Xomat film. Electron microscopy. Electron microscopy of fibroblasts was carried out in the Pathology Department of the Hospital for Sick Children. Cells were fixed with glutaraldehyde, post fixed with osmium tetroxide, dehydrated through graded acetones, infiltrated with a 50:50 mix of absolute acetone and Epon araldite, embedded in pure Epon araldite resin, and polymerized overnight at 607C. Ultrathin sections were cut on a Reichert Ultracut E, collected on copper grids, stained with uranyl acetate and lead citrate, and examined at 60 kV on a Philips 400T transmission electron microscope.
Cell lines. Cells denoted HSC and GM were from The Hospital for Sick Children Cell Culture Service and the NIGMSD Mutant Cell Repository (Camden, NJ), respectively.
Histochemical labeling of mitochondria with Mito Tracker dyes. The use of mitochondrial selective dyes has been described previously (Molecular Probes, Inc., MP 7510 11/19/93 MitoTracker product insert). Fibroblasts were grown on coverslips inside a petri dish filled with a-MEM culture medium. When confluent, the medium was replaced with prewarmed (377C) probe-containing (150 nM M7513 MitoTracker CMXR-H2 , or M7512 CMXR, Molecular Probes, Inc., Eugene, OR) medium, and cells were incubated for 45 min. The medium was replaced with regular medium, and the cells were observed and photographed under a fluorescence microscope.
Isolation of mitochondria from placenta. The mitochondrial fraction from fresh placentae from nor-
Assay of lactate pyruvate ratios. Measurement of lactate/pyruvate ratios in confluent FD and normal
MATERIALS AND METHODS Isolation of DNA. DNA was isolated from normal and FD fibroblasts using standard phenol/chloroform extraction procedures (23).
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fibroblast cultures were carried out as described elsewhere (20). Enzyme activities in cultured skin fibroblasts. Confluent cultures of fibroblasts were removed from culture flasks by mild trypsinization, washed, and sonicated briefly (8-s pulse) using a Sonic dismembrator (Aptek Systems, Farmingham, NY), and measurements were performed on whole cell extracts as follows using a Unicam SP1800 ultraviolet spectrophotometer. Cytochrome oxidase activity was determined as described by DiMauro et al. (25) by following cytochrome c oxidation at 550 nm; succinate cytochrome c reductase was measured by following the reduction of cytochrome c spectrophotometrically at 550 nM (26). A molar extinction coefficient of 0.0185 nmol cm01 min01 was used to determine the amount of nmols of cytochrome c oxidized, or reduced, respectively. RESULTS Mitochondrial DNA In order to assess the overall size and to detect unusual restriction sites in mitochondrial DNA, DNA was first isolated from FD and non-FD cells by standard phenol/chloroform extraction procedures, including ethanol precipitation (23), redissolved in TE, and stored at 0207C. Mitochondria were isolated from human placentae as described under Materials and Methods and the DNA was extracted and linearized as described above and labeled with 32P by random priming as outlined. Southern blots of fibroblast DNA, digested with BamHI, PvuII, or BamHI plus EcoRI, were probed with the purified, 32P-labeled mitochondrial DNA. Data in Fig. 1 indicate that the mitochondrial DNA of FD fibroblasts was of normal length and had no unusual restriction sites, but did contain previously identified BamHI polymorphisms, consistent with BamHI sites at nucleotides 13366 and in the D loop at 16491/16505. Functional Integrity of Fibroblast Mitochondria Cell lines were examined for respiratory chain activity by measuring two respiratory chain components, cytochrome oxidase and succinate cytochrome c reductase. In Table 1 it can be seen that the activities of these two enzyme systems were normal. To further assess this, we followed the intracellular reactions of the fixable mitochondrion-selective dye, mito Tracker CMXR-H2 (see Materials and Meth-
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FIG. 1. Southern blot of mitochondrial DNA. DNA was isolated from FD and normal cells, run in 0.6% agarose gels (2 mg/ lane), transferred to nylon membranes, and probed with BamHI digested, 32P-labeled mitochondrial DNA isolated from human placenta, as described under Materials and Methods. Lane A1, BamHI-digested DNA, FD lines GM05041 (and GM05042, not shown), illustrating the normal BamHI site at nt 14258, and a polymorphic site at 13366, yielding bands of 15.56 and 0.99 kb; lane A2, BamHI or PvuII-digested DNA, FD lines GM02342, GM00850A, normal lines HSC 3781 and 5401 (not all shown), illustrating one cut site only; lane B1, FD line HSC 3616; lane B2, mother of B1, HSC 3654; lane B3, normal line HSC 3781; lane B5, FD lines GM 01777A (and GM02342, not shown). B1, B2, B3, B5 digested with BamHI and EcoRI, showing normal restriction sites for BamHI at nt 14258, and for EcoRII at nts 4121, 5274, 12640, yielding fragments of 7.37, 6.43, 1.83, and 1.15 kb; lane B4, BamHI and EcoRI-digested DNA, FD line GM 02343 (and GM 04634 not shown), with BamHI polymorphism in the D loop at nt 16491/16505, yielding fragments of 7.37, 4.18, 2.25, 1.83, 1.15 kb (38); lane B6, BamHI and EcoRI-digested DNA, FD line GM 05042 (and GM05041, not shown; see lane A1 above), BamHI and EcoRI-digested DNA, with BamHI polymorphism described in lane A1, yielding fragments of 7.37, 6.43, 1.15, 0.94, and 0.89 kb.
ods). When this cell-permeant probe enters an actively respiring cell, it is oxidized to MitoTracker CMXR and sequestered in the mitochondria, where it reacts with thiols on proteins and peptides to form an aldehyde-fixable conjugate, labeling mitochondria with a long-wavelength fluorescence emission.
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TABLE 1 Enzyme Activities in Cultured Skin Fibroblastsa Succinate cytochrome c oxidoreductase (nmol/min/mg)
Cytochrome oxidase (nmol/min/mg)
Cellular lactate/ pyruvate ratio
5.88 { 2.12 (n Å 2) 6.65 { 2.63 (n Å 3) 7.10 { 0.47 (n Å 11)
8.21 { 1.38 (n Å 2) 7.34 { 4.58 (n Å 2) 8.70 { 0.60 (n Å 4)
30.45 { 13.63 (n Å 3) 29.42 { 13.45 (n Å 2) 26.30 { 6.50 (n Å 4)
Cell line Familial dysautonomia Nonfamilial dysautonomia Normal range
Note. Fibroblasts used were as follows: Succinate cytochrome c oxidoreductase (II and III) and cytochrome c oxidase (III), FD, GM 02343, and GM 00850A, non-FD, HSC 3936, 4705, 4212; Lactate/pyruvate ratios, FD, GM 02343, GM 00850A, GM 02342, non-FD, HSC 3979, and 4705. Measurements were carried out as described under Materials and Methods. Values presented in the row entitled ‘‘normal range’’ are data from Dr. B. Robinson’s laboratory at The Hospital for Sick Children, Toronto, Ontario (personal communication). Values represent the mean of duplicate measurements performed on the indicated number of cell lines. a Mean { standard deviation (number of cell lines).
Cells with differing respiratory efficiency will take up this dye differentially. No differences were seen in a time course from 15 to 60 min for the uptake of reduced dye between control and FD fibroblasts. A representative illustration of this is depicted in Fig. 2, taken at 45 min. Mitochondrial Morphology Electron microscopy of fibroblasts did not reveal any unusual morphological features (Fig. 3). Studies with MitoTracker CMXR (M7512), oxidized form, which readily penetrates the mitochondrion, unrelated to the oxidative state (see above), is capable of distinguishing between normal and abnormal structural features of mitochondria. There was no essential difference between FD and non-FD cells (results not shown). In both FD and control fibroblasts mitochondria exhibited the typical bilaminar membranes and tubular cristae. Mitochondria labeled with MitoTracker CMXR occurred in variable numbers and lengths irrespective of whether they were from FD or control fibroblasts. Thus no obvious morphological differences were noted. DISCUSSION The clinical diagnosis of FD is based on five criteria: lack of axon flare after intradermal injection of histamine, absence of fungiform papillae on the tongue, miosis of the pupil after conjunctival injection of metacholine chloride, absent deep tendon reflexes, and diminished tear flow. There are neuropathological findings in the sural nerve, symptoms of ataxia, speech impairment, impaired taste perception, impaired temperature perception, seizures, and
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abnormal EEGs (27). Dysautonomia is often associated with other neuropathies, such as acute sensory motor neuropathy (28), Parkinson’s disease (29), Sandhoff disease (30), and Friedreich’s ataxia (31). Many of the features of FD are progressive, such as the neurogenic-type spinal curvature, renal changes compatible with chronic pyelonephritis, the increase in vomiting, vasomotor crises, and frequency of infections during adolescence (27), deterioration of sensory function, involving both myelinated and unmyelinated axons (32), and the appearance of abnormal brainstem auditory evoked potentials in older patients (33). Previously, we reported an abnormal GSL profile (elevated concentrations of neutral GSL) in FD lymphoblasts and fibroblasts (identical cell lines as used in this study (7)), poor incorporation of glucosamine into lymphoblast GSL (a process requiring ATP) (9), and altered fibroblast plasma membrane morphology (7) (see Introduction). We hypothesized that the increased GSL concentrations could then affect mitochondrial function, as we had previously demonstrated for cerebrosides and lysocerebrosides (see Introduction) and/or that mitochondrial membranes/membrane components (in addition to the plasma membrane) could have been altered. This might have suggested/created possible defects in oxidative phosphorylation (a) related to the etiology of FD, perhaps caused by abnormal lipid profiles or altered/defective interaction of the gene product of DYS on chromosome 9q31 – q33 with mitochondria, or (b) associated with independent accumulation of mitochondrial damage associated with age-related degenerative diseases (17,18).
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FIG. 2. Reaction of MitoTracker CMXR-H2 dye with FD and normal cells. FD fibroblast line GM 02342 (and GM02343, not shown) were grown on coverslips and then incubated in MitoTracker dye (150 nM) for 45 min and observed and photographed under a fluorescence microscope, as described under Materials and Methods. (Top) Non-FD cells, (bottom) FD cells.
In the autosomal dominant form of chronic progressive external ophthalmoplegia, mtDNA rearrangements were identified in various tissues including muscle cells, but excluding leukocytes (34 – 36), due to a nuclear gene defect on chromosome 10q23.3 – q24.3 , predisposing to secondary mtDNA deletions, the mechanism of which re-
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mains elusive. This was associated with pathological changes in the muscle and in the muscle mitochondria themselves. We chose fibroblasts for our study because these cells exhibit abnormal morphology and biochemistry and since autonomic nervous system biopsy material is not normally available from FD patients.
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FIG. 3. Electron micrograph of FD fibroblast line GM 02343, showing normal mitochondrial morphology (see arrows). G, Golgi (40,0001 magnification).
Herein we have described, for FD fibroblasts, which display an altered biochemical and morphological phenotype (7 – 9), normal length mitochondrial DNA with no unusual restriction sites (with the enzymes investigated), normal respiratory chain enzyme activity, and lactate/pyruvate ratios, a normal oxidative state revealed by the CMXR-H2 dye and unremarkable mitochondrial morphology by electron microscopy. Therefore, we conclude that mitochondrial dysfunction, due to GSL accumulation, changes in mitochondrial DNA, or mutation of a chromosome 9q gene involved in mitochondrial function, is neither a primary nor a secondary cause of FD, as determined by a study of FD fibroblasts derived from nonneuronal sites. An electron microscopic investigation of sural nerves in FD children indicated a reduction in the density and total number of
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both myelinated and nonmyelinated axons but gave no evidence of ultrastructural abnormalities (4,37). Since this is a disease of the nervous system, a biochemical defect may exist in neurons or glia which is not overtly morphological and does not affect organization but mitochondrial function. In the absence of viable FD neuronal or glial cultures, this question cannot be answered at this time. ACKNOWLEDGMENTS We are grateful to Ms. Lois Lines for her excellent technical assistance in performing the electron microscopic studies, to Mrs. R. Petrova-Benedict and Ms. T. Myint for carrying out the lactate/ pruvate ratios and respiratory chain enzyme activity measurements, respectively, and to Mrs. T. Selander for her suggestions regarding the Southern blot analysis. This work was supported
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by the Familial Dysautonomia Foundation of Canada and the Medical Research Council of Canada.
REFERENCES 1. Axelrod F, Pearson J. Congenital sensory neuropathies. Am J Dis Child 138:947–954, 1984. 2. Maayan C, Kaplan E, Shachan S, Peleg O, Godfrey S. Incidence of familial dysautonomia in Israel 1977–1981. Clin Genet 32:106–108, 1987. 3. Riley CM, Day RL, Greely DM, Langford WS. Central autonomic dysfunction with defective lacrimation. Pediatrics 3:468–478, 1949. 4. Aguayo AJ, Cherunada PV, Nair MB, Bray GM. Peripheral nerve abnormalities in the Riley-Day syndrome. Arch Neurol 24:106–116, 1971. 5. McKusick VA, Norum RA, Farkas HJ, Brunt PW, Mahloudji M. The Riley–Day syndrome—Observations on genetics and survivorship. Isr J Med Sci 3:372–379, 1967. 6. Slaugenhaupt SA, Liebert CB, Mull J, MacCormack K, LeBel A, O’Leary K, Lucente D, McCormick MK, Buckler AJ, Maayan C, Axelrod F, Blumenfeld A, Gusella JF. Evaluation of candidate genes for Familial Dysautonomia. Am J Hum Genet 57:A270, 1995. 7. Strasberg PM, Yeger H, Warren I. Increased globotriaosylceramide in familial dysautonomia. Lipids 27:978–983, 1992. 8. Pereira J, Boyd B, Newbigging J, Lingwood C, Strasberg PM. Increased globotriaosylceramide on plasma membranes of synchronized Familial Dysautonomia cells. J Mol Neurosci 5:1–10, 1994. 9. Strasberg PM, Novak A, Warren IB. Decreased incorporation of D-glucosamine into glycosphingolipids of intact Familial Dysautonomia lymphoblasts. J Mol Neurosci 6:121– 130, 1995. 10. Strasberg PM. Cerebrosides and psychosine disrupt mitochondrial functions. Can J Biochem Cell Physiol 64:485– 489, 1986. 11. Strasberg PM, Callahan JW. Lysosphingolipids and mitochondrial function. II. Deleterious effects of sphingophosphorylcholine. Can J Biochem 66:1322–1332, 1988. 12. Strasberg PM, Callahan JW. Psychosine and sphingosylphosphorylcholine bind to mitochondrial membranes and disrupt their function. In Lipid Storage Disorders (Salvayre, R, Douste-Blazy, L, Gatt, S, Eds.). New York: Plenum, 1988, pp 601–606. 13. Conradi NG, Sourander P, Nilsson O, Svennerholm L, Erickson A. Neuropathology of the Norbottnian type of Gaucher disease. Morphological and biochemical studies. Acta Neuropathol 65:99–109, 1984. 14. Hakomori S-I. Bifunctional role of glycosphingolipids. J Biol Chem 265:18713–18716, 1990. 15. Chao MV. Ceramide: A potential second messenger in the nervous system. Mol Cell Neurosci 6:91–96, 1995. 16. Obeid LM, Hannun YA. Ceramide: A stress signal and mediator of growth suppression and apoptosis. J Cell Biochem 58:191–198, 1995.
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17. Wallace DC. Mitochondrial genetics: A paradigm for aging and degenerative diseases? Science 256:628–632, 1992. 18. Wallace DC. Mitochondrial DNA variation in human evolution, degenerative diseases, and aging. Am J Hum Genet 57:201–221, 1995. 19. Wisniewski KE, Kaczmarski W, Golabek AA, Kida E. Rapid detection of subunit c of mitochondrial ATP synthase in urine as a diagnostic screening method for neuronal ceroidlipofuscinoses. Am J Med Genet 57:246–249, 1995. 20. Robinson BH, MacKay N, Goodyer P, Lancaster G. Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia. Am J Hum Genet 37:938–946, 1985. 21. Goto Y, Nonaka I, Horai S. A mutation in the tRNAleu(UUR) gene associated with MELAS subgroup of mitochondrial encephalopathies. Nature 348:651–653, 1990. 22. Shoffner JM, Lott MT, Lezza AMS, Seibel P, Ballinger SW, Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNAlys mutation. Cell 61:931–937, 1990. 23. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2nd ed., 1989. 24. Moore CL, Strasberg PM, Kovac C. Studies on mitochondrial creatine-phosphokinase: Rat skeletal muscle mitochondria. Tex Rep Biol Med 31:367–384, 1973. 25. DiMauro S, Nicholson JF, Hays AP, Eastwood AB, Papadimitriou A, Koeningsberger R, DeVivo DC. Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency. Ann Neurol 14:226–234, 1983. 26. Fischer JC, Ruitenbeek W, Stadhouders AM, Trijbels FJM, Sengers RCA, Janssen AJM, Veercamp JH. Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin Chim Acta 145:89–94, 1985. 27. Axelrod FB, Nachtigal R, Dancis J. Familial dysautonomia: Diagnosis, pathogenesis and management. Adv Pediatr 21:75–96, 1974. 28. Yokota T, Hayashi M, Hirashima F, Mitani M, Tanabe H, Tsukagoshi H. Dysautonomia with acute sensory motor neuropathy. A new classification of acute autonomic neuropathy. Arch Neurol 51:1022–1031, 1994. 29. Sandyk R, Awerbuch GI. Dysautonomia in Parkinson’s disease: Relationship to motor disability. Int J Neurosci 64:23– 31, 1992. 30. Modigliani R, Lemann M, Melancon SB, Mikol J, Potier M, Salmeron M, Said G, Poitras P. Diarrhea and autonomic dysfunction in a patient with hexosaminidase B deficiency (Sandhoff disease). Gastroenterology 106:775–781, 1994. 31. Margalith D, Dunn HG, Carter JE, Wright JM. Friedreich’s ataxia with dysantonomia and labile hypertension. Can J Neurol Sci 11:73–95, 1984. 32. Axelrod FB, Kamla L, Fish I, Pearson J, Sein MA, Speilholz N. Progressive sensory loss in familial dysautonomia. Pediatrics 67:517–522, 1981. 33. Lahat E, Aladjem M, Mor A, Azizi E, Arlazarof A. Brainstem auditory evoked potentials in familial dysautonomia. Dev Med Child Neurol 34:690–693, 1992. 34. Kaukonen JA, Amaati P, Suomalainen A, Rotig A, Piscaglia
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senbach J, Zeviano M, Somer H, Peltonen L. An autosomal locus predisposing to deletions of mitochondrial DNA. Nature Genet 9:146–151, 1995. 37. Pearson J, Dancis J, Axelrod F, Grover N. The sural nerve in familial dysautonomia. J Neuropathol Exp Neurol 34:413–424, 1975. 38. Bonne-Tamir B, Johnson MJ, Natali A, Wallace DC, CavalliSforza LL. Human mitochondrial DNA types in two Israeli populations—A comparative study at the DNA level. Am J Hum Genet 38:341–351, 1986.
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Normal Mitochondrial DNA and Respiratory Chain Activity in Familial Dysautonomia Fibroblasts PAULA STRASBERG,*,†,‡ PHILLIPA BRIDGE,* FRANK MERANTE,§ HERMAN YEGER,Ø
AND
JUDE PEREIRA*
*Division of Neuroscience, The Research Institute, and †Department of Clinical Genetics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; ‡Department of Clinical Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; §Center for Cardiovascular Research, General Division, The Toronto Hospital, Toronto, Ontario, Canada; and Ø Department of Pathology, The Hospital for Sick Children, and The University of Toronto, Toronto, Ontario, Canada Received November 7, 1995, and in revised form March 29, 1996
III), with features of absent lingual fungiform papillae, lack of tearing, vasomotor instability, increased sweating, cold hands and feet, corneal anesthesia, neurological defects, respiratory complications, erythematous blotching of the skin, and scoliosis (1–3), restricted to Ashkenazi Jews. In sural nerve, there is a decrease in the number of myelinated fibers (4). Pathognomic for FD is the fact that FD patients do not exhibit the wheal and flare response to intradermally injected histamine (5). The gene (DYS) maps to chromosome 9q31 (6) (6–10). Previously, we found increases in Gb3 in FD fibroblasts and lymphoblasts and a decrease in ganglioside levels, as well as light microscopic pleiomorphic changes in FD fibroblasts, suggestive of changes in the plasma membrane (7). We described an increase in Gb3 on the surface of synchronized cells at the G1/ S boundary of the cell cycle, based on Gb3 –verotoxin (derived from E. coli) interactions (8). Using D-glucosamine-1-14C as an in vitro precursor, we reported a marked decrease in the rate of incorporation of Dglucosamine into the sialic acid and the N-acetylgalacto/glucosamine moieties of gangliosides and neutral GSL in intact FD compared to control lymphoblasts. The total ganglioside content of FD lymphoblasts (primarily GM3 , measured as incorporation of 3H from NaB3H4) was also decreased. These data indicated differences in the turnover of sialic acid and N-acetylated sugar constituents in FD versus normal cells (9). Earlier experiments in our laboratory demonstrated that cerebrosides and lysocerebrosides, at concentrations approaching those in lysosomal stor-
Familial dysautonomia (FD), an autosomal recessive disease mapped to chromosome 9q31, is a sensory and autonomic neuropathy of unknown etiology. We have previously reported light microscopic pleiomorphic changes in cells suggestive of altered plasma membranes, an increase in globotriaosylceramide (Gb3), reflected by an increase in Gb3 on the surface of the plasma membrane, and a decrease in the rate and amount of ganglioside synthesized. In unrelated studies, we demonstrated that storage of glycospingolipids (GSL) is deleterious to mitochondrial function. Recently, mitochondrial dysfunction has been associated with neurodegenerative disease, superimposed on an autosomal inheritance pattern. We have now probed Southern blots of total FD fibroblast DNA, digested with BamHI, EcoRII, and/or PvuII, with purified placental 32P-labeled mitochondrial DNA. The sizes of all FD mitochondrial DNAs were normal (16,569 bp), some containing previously identified BamHI polymorphisms. Lactate/ pyruvate ratios, and activities of Complexes II and III, matched those of control cells. Electron microscopy revealed morphologically normal mitochondria, in conjunction with a normal oxidative state, determined using the redox dyes Mito Tracker CMXR and CMXR-H2 and fluorescence microscopy. We conclude that mitochondrial dysfunction, due to GSL accumulation, changes in mitochondrial DNA, or mutation of a chromosome 9q gene involved in mitochondrial function, is neither a primary nor a secondary cause of FD, as determined by a study of FD fibroblasts. q 1996 Academic Press, Inc.
Familial dysautonomia (FD, MIM 223900) is a hereditary sensory and autonomic neuropathy (HSAN20 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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age diseases (LSD), inhibited oxidative phorphorylation and caused mitochondrial swelling in a manner suggestive of distortion of the mitochondrial membranes (10–12). Furthermore, it is generally accepted that there is a loss of neurons and myelin in the vicinity of accumulations of storage cells (13). It has more recently been shown that GSL or their modified catabolites modulate transmembrane signal transduction at the cell surface, regulate cell proliferation, induce differentiation, and serve as receptors for cellular interactions, bacterial toxins, and hormones (14). Ceramide has been cited as a potential second messenger in the nervous system (15) and as a stress signal and mediator of growth suppression and apoptosis (16). Recently, mitochondrial dysfunction has been associated with neurodegenerative disease, superimposed on an autosomal inheritance pattern, for example, in Alzheimer and Parkinson diseases, and diabetes mellitus, due to mitochondrial DNA mutagenesis from oxygen radical generation (17,18). In the case of Batten disease, there is accumulation of subunit c of the mitochondrial ATP synthase, a protein derived from nuclear DNA (19). This may not be the primary event in the etiology of Batten disease, but nonetheless represents a form of mitochondrial dysfunction associated with a genetic disease. Our studies previously indicated alterations in GSL in FD with the potentially deleterious effects on mitochondria noted above. Normally glucosamine is incorporated into gangliosides as CMP-sialic acid and high-energy UDP sugars, both of which cannot be synthesized without ATP. Since alterations in mitochondrial function have been a primary cause of (e.g., neonatal lacticacidemia (20), MELAS (21), MERRF (22)), or a secondarily related finding in, other neurological diseases, we decided to investigate mitochondrial function and the integrity of mitochondrial DNA in FD.
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mal births was isolated and purified using a Ficoll gradient as described elsewhere (24). DNA was extracted from the mitochondria as above. Random priming of mitochondrial DNA. Mitochondrial DNA was linearized by digestion with BamHI, reprecipitated, taken up in TE (10 mM Tris– HCl, 1 mM EDTA, pH 7.4) (23), and labeled with 32P by random priming using the Boehringer Mannheim random primed kit (Model 1004760) according to manufacturer’s instructions. Southern blotting of fibroblast DNA. Two micrograms of DNA per sample was digested with BamHI, PvuII, or BamHI plus EcoRI overnight (in a volume of 200 ml), precipitated, redissolved, and loaded with tracking dye onto 0.6% agarose gels, along with appropriate size standards. Gel electrophoresis and Southern transfer were done according to standard procedures (23). Blots were hybridized with the 32Pmitochondrial DNA (2.5 1 106 cpm per ml, 10 ml per blot), washed with 0.5 1 SSPE (23), 0.1% SDS for 30 min at 427C and 15 min at 657C, and exposed to Kodak Xomat film. Electron microscopy. Electron microscopy of fibroblasts was carried out in the Pathology Department of the Hospital for Sick Children. Cells were fixed with glutaraldehyde, post fixed with osmium tetroxide, dehydrated through graded acetones, infiltrated with a 50:50 mix of absolute acetone and Epon araldite, embedded in pure Epon araldite resin, and polymerized overnight at 607C. Ultrathin sections were cut on a Reichert Ultracut E, collected on copper grids, stained with uranyl acetate and lead citrate, and examined at 60 kV on a Philips 400T transmission electron microscope.
Cell lines. Cells denoted HSC and GM were from The Hospital for Sick Children Cell Culture Service and the NIGMSD Mutant Cell Repository (Camden, NJ), respectively.
Histochemical labeling of mitochondria with Mito Tracker dyes. The use of mitochondrial selective dyes has been described previously (Molecular Probes, Inc., MP 7510 11/19/93 MitoTracker product insert). Fibroblasts were grown on coverslips inside a petri dish filled with a-MEM culture medium. When confluent, the medium was replaced with prewarmed (377C) probe-containing (150 nM M7513 MitoTracker CMXR-H2 , or M7512 CMXR, Molecular Probes, Inc., Eugene, OR) medium, and cells were incubated for 45 min. The medium was replaced with regular medium, and the cells were observed and photographed under a fluorescence microscope.
Isolation of mitochondria from placenta. The mitochondrial fraction from fresh placentae from nor-
Assay of lactate pyruvate ratios. Measurement of lactate/pyruvate ratios in confluent FD and normal
MATERIALS AND METHODS Isolation of DNA. DNA was isolated from normal and FD fibroblasts using standard phenol/chloroform extraction procedures (23).
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fibroblast cultures were carried out as described elsewhere (20). Enzyme activities in cultured skin fibroblasts. Confluent cultures of fibroblasts were removed from culture flasks by mild trypsinization, washed, and sonicated briefly (8-s pulse) using a Sonic dismembrator (Aptek Systems, Farmingham, NY), and measurements were performed on whole cell extracts as follows using a Unicam SP1800 ultraviolet spectrophotometer. Cytochrome oxidase activity was determined as described by DiMauro et al. (25) by following cytochrome c oxidation at 550 nm; succinate cytochrome c reductase was measured by following the reduction of cytochrome c spectrophotometrically at 550 nM (26). A molar extinction coefficient of 0.0185 nmol cm01 min01 was used to determine the amount of nmols of cytochrome c oxidized, or reduced, respectively. RESULTS Mitochondrial DNA In order to assess the overall size and to detect unusual restriction sites in mitochondrial DNA, DNA was first isolated from FD and non-FD cells by standard phenol/chloroform extraction procedures, including ethanol precipitation (23), redissolved in TE, and stored at 0207C. Mitochondria were isolated from human placentae as described under Materials and Methods and the DNA was extracted and linearized as described above and labeled with 32P by random priming as outlined. Southern blots of fibroblast DNA, digested with BamHI, PvuII, or BamHI plus EcoRI, were probed with the purified, 32P-labeled mitochondrial DNA. Data in Fig. 1 indicate that the mitochondrial DNA of FD fibroblasts was of normal length and had no unusual restriction sites, but did contain previously identified BamHI polymorphisms, consistent with BamHI sites at nucleotides 13366 and in the D loop at 16491/16505. Functional Integrity of Fibroblast Mitochondria Cell lines were examined for respiratory chain activity by measuring two respiratory chain components, cytochrome oxidase and succinate cytochrome c reductase. In Table 1 it can be seen that the activities of these two enzyme systems were normal. To further assess this, we followed the intracellular reactions of the fixable mitochondrion-selective dye, mito Tracker CMXR-H2 (see Materials and Meth-
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FIG. 1. Southern blot of mitochondrial DNA. DNA was isolated from FD and normal cells, run in 0.6% agarose gels (2 mg/ lane), transferred to nylon membranes, and probed with BamHI digested, 32P-labeled mitochondrial DNA isolated from human placenta, as described under Materials and Methods. Lane A1, BamHI-digested DNA, FD lines GM05041 (and GM05042, not shown), illustrating the normal BamHI site at nt 14258, and a polymorphic site at 13366, yielding bands of 15.56 and 0.99 kb; lane A2, BamHI or PvuII-digested DNA, FD lines GM02342, GM00850A, normal lines HSC 3781 and 5401 (not all shown), illustrating one cut site only; lane B1, FD line HSC 3616; lane B2, mother of B1, HSC 3654; lane B3, normal line HSC 3781; lane B5, FD lines GM 01777A (and GM02342, not shown). B1, B2, B3, B5 digested with BamHI and EcoRI, showing normal restriction sites for BamHI at nt 14258, and for EcoRII at nts 4121, 5274, 12640, yielding fragments of 7.37, 6.43, 1.83, and 1.15 kb; lane B4, BamHI and EcoRI-digested DNA, FD line GM 02343 (and GM 04634 not shown), with BamHI polymorphism in the D loop at nt 16491/16505, yielding fragments of 7.37, 4.18, 2.25, 1.83, 1.15 kb (38); lane B6, BamHI and EcoRI-digested DNA, FD line GM 05042 (and GM05041, not shown; see lane A1 above), BamHI and EcoRI-digested DNA, with BamHI polymorphism described in lane A1, yielding fragments of 7.37, 6.43, 1.15, 0.94, and 0.89 kb.
ods). When this cell-permeant probe enters an actively respiring cell, it is oxidized to MitoTracker CMXR and sequestered in the mitochondria, where it reacts with thiols on proteins and peptides to form an aldehyde-fixable conjugate, labeling mitochondria with a long-wavelength fluorescence emission.
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TABLE 1 Enzyme Activities in Cultured Skin Fibroblastsa Succinate cytochrome c oxidoreductase (nmol/min/mg)
Cytochrome oxidase (nmol/min/mg)
Cellular lactate/ pyruvate ratio
5.88 { 2.12 (n Å 2) 6.65 { 2.63 (n Å 3) 7.10 { 0.47 (n Å 11)
8.21 { 1.38 (n Å 2) 7.34 { 4.58 (n Å 2) 8.70 { 0.60 (n Å 4)
30.45 { 13.63 (n Å 3) 29.42 { 13.45 (n Å 2) 26.30 { 6.50 (n Å 4)
Cell line Familial dysautonomia Nonfamilial dysautonomia Normal range
Note. Fibroblasts used were as follows: Succinate cytochrome c oxidoreductase (II and III) and cytochrome c oxidase (III), FD, GM 02343, and GM 00850A, non-FD, HSC 3936, 4705, 4212; Lactate/pyruvate ratios, FD, GM 02343, GM 00850A, GM 02342, non-FD, HSC 3979, and 4705. Measurements were carried out as described under Materials and Methods. Values presented in the row entitled ‘‘normal range’’ are data from Dr. B. Robinson’s laboratory at The Hospital for Sick Children, Toronto, Ontario (personal communication). Values represent the mean of duplicate measurements performed on the indicated number of cell lines. a Mean { standard deviation (number of cell lines).
Cells with differing respiratory efficiency will take up this dye differentially. No differences were seen in a time course from 15 to 60 min for the uptake of reduced dye between control and FD fibroblasts. A representative illustration of this is depicted in Fig. 2, taken at 45 min. Mitochondrial Morphology Electron microscopy of fibroblasts did not reveal any unusual morphological features (Fig. 3). Studies with MitoTracker CMXR (M7512), oxidized form, which readily penetrates the mitochondrion, unrelated to the oxidative state (see above), is capable of distinguishing between normal and abnormal structural features of mitochondria. There was no essential difference between FD and non-FD cells (results not shown). In both FD and control fibroblasts mitochondria exhibited the typical bilaminar membranes and tubular cristae. Mitochondria labeled with MitoTracker CMXR occurred in variable numbers and lengths irrespective of whether they were from FD or control fibroblasts. Thus no obvious morphological differences were noted. DISCUSSION The clinical diagnosis of FD is based on five criteria: lack of axon flare after intradermal injection of histamine, absence of fungiform papillae on the tongue, miosis of the pupil after conjunctival injection of metacholine chloride, absent deep tendon reflexes, and diminished tear flow. There are neuropathological findings in the sural nerve, symptoms of ataxia, speech impairment, impaired taste perception, impaired temperature perception, seizures, and
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abnormal EEGs (27). Dysautonomia is often associated with other neuropathies, such as acute sensory motor neuropathy (28), Parkinson’s disease (29), Sandhoff disease (30), and Friedreich’s ataxia (31). Many of the features of FD are progressive, such as the neurogenic-type spinal curvature, renal changes compatible with chronic pyelonephritis, the increase in vomiting, vasomotor crises, and frequency of infections during adolescence (27), deterioration of sensory function, involving both myelinated and unmyelinated axons (32), and the appearance of abnormal brainstem auditory evoked potentials in older patients (33). Previously, we reported an abnormal GSL profile (elevated concentrations of neutral GSL) in FD lymphoblasts and fibroblasts (identical cell lines as used in this study (7)), poor incorporation of glucosamine into lymphoblast GSL (a process requiring ATP) (9), and altered fibroblast plasma membrane morphology (7) (see Introduction). We hypothesized that the increased GSL concentrations could then affect mitochondrial function, as we had previously demonstrated for cerebrosides and lysocerebrosides (see Introduction) and/or that mitochondrial membranes/membrane components (in addition to the plasma membrane) could have been altered. This might have suggested/created possible defects in oxidative phosphorylation (a) related to the etiology of FD, perhaps caused by abnormal lipid profiles or altered/defective interaction of the gene product of DYS on chromosome 9q31 – q33 with mitochondria, or (b) associated with independent accumulation of mitochondrial damage associated with age-related degenerative diseases (17,18).
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FIG. 2. Reaction of MitoTracker CMXR-H2 dye with FD and normal cells. FD fibroblast line GM 02342 (and GM02343, not shown) were grown on coverslips and then incubated in MitoTracker dye (150 nM) for 45 min and observed and photographed under a fluorescence microscope, as described under Materials and Methods. (Top) Non-FD cells, (bottom) FD cells.
In the autosomal dominant form of chronic progressive external ophthalmoplegia, mtDNA rearrangements were identified in various tissues including muscle cells, but excluding leukocytes (34 – 36), due to a nuclear gene defect on chromosome 10q23.3 – q24.3 , predisposing to secondary mtDNA deletions, the mechanism of which re-
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mains elusive. This was associated with pathological changes in the muscle and in the muscle mitochondria themselves. We chose fibroblasts for our study because these cells exhibit abnormal morphology and biochemistry and since autonomic nervous system biopsy material is not normally available from FD patients.
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FIG. 3. Electron micrograph of FD fibroblast line GM 02343, showing normal mitochondrial morphology (see arrows). G, Golgi (40,0001 magnification).
Herein we have described, for FD fibroblasts, which display an altered biochemical and morphological phenotype (7 – 9), normal length mitochondrial DNA with no unusual restriction sites (with the enzymes investigated), normal respiratory chain enzyme activity, and lactate/pyruvate ratios, a normal oxidative state revealed by the CMXR-H2 dye and unremarkable mitochondrial morphology by electron microscopy. Therefore, we conclude that mitochondrial dysfunction, due to GSL accumulation, changes in mitochondrial DNA, or mutation of a chromosome 9q gene involved in mitochondrial function, is neither a primary nor a secondary cause of FD, as determined by a study of FD fibroblasts derived from nonneuronal sites. An electron microscopic investigation of sural nerves in FD children indicated a reduction in the density and total number of
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both myelinated and nonmyelinated axons but gave no evidence of ultrastructural abnormalities (4,37). Since this is a disease of the nervous system, a biochemical defect may exist in neurons or glia which is not overtly morphological and does not affect organization but mitochondrial function. In the absence of viable FD neuronal or glial cultures, this question cannot be answered at this time. ACKNOWLEDGMENTS We are grateful to Ms. Lois Lines for her excellent technical assistance in performing the electron microscopic studies, to Mrs. R. Petrova-Benedict and Ms. T. Myint for carrying out the lactate/ pruvate ratios and respiratory chain enzyme activity measurements, respectively, and to Mrs. T. Selander for her suggestions regarding the Southern blot analysis. This work was supported
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by the Familial Dysautonomia Foundation of Canada and the Medical Research Council of Canada.
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