Abstract
The purpose of this study was to examine mitochondrial changes in the spinal cord of transgenic mice of a relatively low transgenic copy number (gene copy 10) expressing a G93A mutant human Cu/Zn superoxide dismutase (SOD1) that were generated in our own laboratories by electron and immunoelectron microscopy from presymptomatic to symptomatic stages. Age-matched non-transgenic mice served as controls at each stage. Ultrastructurally, at the early presymptomatic stage, many mitochondria in large myelinated axons exhibited swelling with an increased number of cristae, and bore small vacuoles in the matrix, cristae or both, in the anterior root exit zone, anterior root, and in the neuropils of the ventral portion of the anterior horn. At the late presymptomatic stage, vacuoles of various sizes (including large ones) were observed in the same regions as in the previous stage. The intermembrane space of mitochondria was also vacuolated. In mitochondria with advanced vacuolation, the vacuolar space was filled with a granular or amorphous substance. At the symptomatic stage, mitochondrial vacuolation seen in the late presymptomatic stage persisted, although to a lesser extent. These vacuolated mitochondria were predominantly seen in the axons, but not in the somata of normal-looking neurons or dendrites at any stage, which differs from that described in other reports. Non-transgenic littermates occasionally exhibited vacuolar changes in the axons of anterior horns. However, they were smaller both in size and number than those in transgenic mice. By immunoelectron microscopy using an immunogold labeling method, at the presymptomatic and symptomatic stages both SOD1 and ubiquitin determinants were localized in vacuolated mitochondria, particularly in the granular or amorphous substance of large vacuoles, but were not detected in most normal-appearing mitochondria. The SOD1-immunoreactive mitochondria were exclusively observed in the axons, and not in proximal dendrites or somata. These findings suggest that the toxicity of mutant SOD1 directly affects mitochondria in the axons and increases with the disease progression. Thus, the mutant SOD1 toxicity might disrupt axonal transport of substrates needed for neuronal viability, leading to motor neuron degeneration. The localization of both ubiquitin and SOD1 in vacuolated mitochondria indicates that protein degradation by ubiquitin-proteasomal system may be also disrupted by several pathomechanisms, such as decreased processing of ubiquitinated proteins due to impairment of mitochondrial function or of proteasomal function, both of which are caused by mutant SOD1. Moreover, giant mitochondrial vacuoles occupying almost the entire axonal caliber could be another contributing factor in motor neuron degeneration, in that they could physically block axonal transport.
Similar content being viewed by others
References
Andreassen OA, Ferrante RJ, Klivenyi P, Klein AM, Shinobu LA, Epstein CJ, Beal MF (2000) Partial deficiency of manganese superoxide dismutase exacerbates a transgenic mouse model of amyotrophic lateral sclerosis. Ann Neurol 47:447–455
Andreassen OA, Ferrante RJ, Klivenyi P, Klein AM, Dedeoglu A, Albers DS, Kowell NW, Beal MF (2001) Transgenic ALS mice show increased vulnerability to the mitochondrial toxins MPTP and 3-nitropropionic acid. Exp Neurol 168:356–363
Asayama K, Janco RL, Burr IM (1985) Selective induction of manganous superoxide dismutase in human monocytes. Am J Physiol 249:C393–C397
Beal MF (1992) Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol 1:119–130
Borthwick GM, Johnson MA, Ince PG, Shaw PJ, Turnbull DM (1999) Mitochondrial enzyme in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death. Ann Neurol 46:787–790
Bowling AC, Schulz JB, Brown RH, Beal MF (1993) SOD activity, oxidative damage, and mitochondrial energy metabolism in familial and sporadic ALS. J Neurochem 61:2322–2325
Browne SE, Bowling AC, Baik MJ, Gurney M, Brown RH Jr, Beal MF (1998) Metabolic dysfunction in familial, but not sporadic, amyotrophic lateral sclerosis. J Neurochem 71:281–287
Carri MT, Ferri A, Battistoni A, Famhy L, Cabbianelli R, Poccia F, Rotilio G (1997) Expression of a Cu, Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cyotosolic Ca2+ concentration in transfected neuroblastoma SH-Sy5Y cells. FEBS Lett 414:365–368
Comi GP, Bordoni A, Salani S, Franceschina L, Sciacco M, Prelle A, Fortunato F, Zeviani M, Napoli L, Bresolin N, Moggio M, Ausenda CD, Taanman J-W, Scarlato G (1998) Cytochrome c oxidase subunit I microdeletion in a patient with motor neuron disease. Ann Neurol 43:110–116
Crapo JD, Oury T, Rabouille C, Slot JW, Chang LY (1992) Copper, zinc superoxide dismutase in primarily a cytosolic protein in human cells. Proc Natl Acad Sci USA 89:10405–10409
Curti D, Malaspina A, Facchetti G, Camana C, Mazzini L, Tosca P, Zerbi F, Ceroni M (1996) Amyotrophic lateral sclerosis: oxidative energy metabolism and calcium homeostasis in peripheral blood lymphocytes. Neurology 47:1060–1064
Dal Canto MC, Gurney ME (1994) Development of central nervous system pathology in a murine transgenic model of human amyotrophic lateral sclerosis. Am J Pathol 145:1271–1280
Dal Canto MC, Gurney ME (1995) Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu, Zn SOD, and in mice overexpressing wild-type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res 676:25–40
Dhaliwal GK, Grewal RP (2000) Mitochondrial DNA deletion mutation levels are elevated in ALS brains. Neuroreport 11:2507–2509
Estevez AG, Crow JP, Sampson JB, Reiter C, Zhuang YX, Richardson GJ, Tarpey MM, Barbeito L, Beckman JS (1999) Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase. Science 286:2498–2500
Fujita K, Yamauchi M, ShibayamaK, Ando M, Honda M, Nagata Y (1996) Decreased cytochrome c oxidase activity but unchanged superoxide dismutase and glutathione peroxidase activities in the spinal cords of patients with amyotrophic lateral sclerosis. J Neurosci Res 45:276–281
Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, Caliendo J, Hentati A, Kwon YW, Deng HX, et al (1994) Motor neuron degeneration in mice that express a human Cu/Zn superoxide dismutase mutation. Science 264:1772–1775
Higgins CMJ, Jung C, Ding H, Xu Z (2002) Mutant Cu, Zn superoxide dismutase that causes motoneuron degeneration is present in mitochondria in the CNS. J Neurosci 22:RC215
Ince P, Stout N, Shaw P, Slade J, Hunziker W, Heizmann CW, Baimbridge KG (1993) Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease. Neuropathol Applied Neurobiol 19:291–299
Jaarsma D, Rognoni F, Duijn W van, Verspaget HW, Haasdijk ED, Holstege JC (2001) CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations. Acta Neuropathol 102:293–305
Jung C, Higgins CMJ, Xu Z (2002) Mitochondrial electron transport chain complex dysfunction in a transgenic mouse model for amyotrophic lateral sclerosis. J Neurochem 3:535–545
Kaal EC, Vlug AS, Versleijen MW, Kuilman M, Joosten EA, Bar PR (2000) Chronic mitochondrial inhibition induces selective motoneuron death in vitro: a new model for amyotrophic lateral sclerosis. J Neurochem 74:1158–1165
Kong J, Xu Z (1998) Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J Neurosci 18:3241–3250
Levine JB, Kong J, Nadler M, Xu Z (1999) Astrocytes interact intimately with degenerating motor neurons in mouse amyotrophic lateral sclerosis (ALS). Glia 28:215–224
Masui Y, Mozai T, Kakehi K (1985) Functional and morphometric study of the liver in motor neuron disease. J Neurol 232:15–19
Mattiazzi M, D’Aurelio M, Gajewski CD, Martushova K, Kiaei M, Beal MF, Manfredi G (2002) Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice. J Biol Chem 277:29626–29633
McLaughlin BA, Nelson D, Silver IA, Erecinska M, Chesselet MF (1998) Methylmalonate toxicity in primary neuronal cultures. Neuroscience 86:279–290
Menzies FM, Cookson MR, Taylor RW, Turnbull DM, Chrzanowska-Lightowlers ZMA, Dong L, Figlewicz DA, Shaw PJ (2002) Mitochondrial dysfunction in a cell culture model of familial amyotrophic lateral sclerosis. Brain 125:1522–1533
Nakano K, Hirayama K, Terao K (1987) Hepatic ultrastructural changes and liver dysfunction in amyotrophic lateral sclerosis. Arch Neurol 44:103–106
Okado-Matsumoto A, Fridovich I (2001) Subcellular distribution of superoxide dismutase (SOD) in rat liver: Cu, Zn-SOD in mitochondria. J Biol Chem 276:38388–38393
Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O’Regan JP, Deng HX, et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59–62
Sasaki S, Iwata M (1996) Impairment of fast axonal transport in the proximal axons of anterior horn neurons in amyotrophic lateral sclerosis. Neurology 47:535–540
Sasaki S, Iwata M (1996) Ultrastructural study of synapses in the anterior horn neurons of patients with amyotrophic lateral sclerosis. Neurosci Lett 204:53–56
Siklos L, Engelhardt J, Harati Y, Smith RG, Joo F, Appel SH (1996) Ultrastructural evidence for altered calcium in motor nerve terminals in amyotrophic lateral sclerosis. Ann Neurol 39:203–216
Slot JW, Geuze HJ, Freeman BA, Crapo JD (1986) Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parenchymal cells. Lab Invest 55:363–571
Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001) A fraction of yeast Cu, Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. a physiological role for SOD1 in guarding against mitochondrial oxidative damage. J Biol Chem 276:38084–38089
Weisiger RA, Fridovich I (1973) Mitochondrial superoxide dismutase. Site of synthesis and intramitochondrial localization. J Biol Chem 248:4793–4796
Wiedau-Pazos M, Goto JJ, Rabizadch S, Gralla EB, Roc JA, Lee MK, Valentine JS, Bredesen DE (1996) Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 271:515–518
Wiedemann FR, Manfredi G, Mawrin C, Beal MF, Schon EA (2002) Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients. J Neurochem 80:616–625
Wong PC, Pardo CA, Borchelt DR, Lee MK, Copeland NG, Jenkins NA, Sisodia SS, Cleveland DW, Price DL (1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14:1105–1116
Xu ZS, Higgins CMJ (2002) Mechanism of mitochondrial vacuolation in a transgenic mouse model for ALS. Amyotroph Lateral Scler other Motor Neuron Disord 3 (Suppl 2):26–27
Acknowledgements
This work was supported by a Grant-in-Aid for General Scientific Research (C) from the Japanese Ministry of Education, Science and Culture, and by a grant from the Japan ALS Association. We gratefully acknowledge the technical assistance of Dr. N. Shibata and Mr. M. Karita (Department of Pathology, Tokyo Women’s Medical University, Tokyo) in immunoelectron microscopy.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sasaki, S., Warita, H., Murakami, T. et al. Ultrastructural study of mitochondria in the spinal cord of transgenic mice with a G93A mutant SOD1 gene. Acta Neuropathol 107, 461–474 (2004). https://doi.org/10.1007/s00401-004-0837-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00401-004-0837-z