- Email: [email protected]
Change in superoxide dismutase1 protein localization towards mitochondria: an immunohistochemical study in transgenic G93A mice
Neuroscience Letters 332 (2002) 53–56 www.elsevier.com/locate/neulet
Change in superoxide dismutase1 protein localization towards mitochondria: an immunohistochemical study in transgenic G93A mice Hristelina Ilieva, Isao Nagano, Tetsuro Murakami, Mito Shiote, Yasuhiro Manabe, Koji Abe* Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 1 July 2002; received in revised form 10 August 2002; accepted 12 August 2002
Abstract Localization of superoxide dismutase1 (SOD1) and mitochondrial glucose-regulated protein 75 (Grp75), were examined in the spinal cords of transgenic (Tg) mice expressing human mutant SOD1 protein (G93A) and wild-type (Wt) controls at 8, 20 and 32 weeks. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons. Colocalization of Grp75 and SOD1 signals was demonstrated in Wt and presymptomatic Tg animals, while it was lost in Tg mice at a symptomatic age. The present results suggest that loss of SOD1 protein from mitochondria could contribute to motor neuron damage. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Immunohistochemistry; Mitochondria; Superoxide dismutase1
Amyotrophic lateral sclerosis is a fatal neurological disease characterized by selective degeneration of upper and lower motor neurons. About 5–10% of all cases are familial 20% of which are results of dominant mutations in the gene encoding a ubiquitously expressed Cu/Zn superoxide dismutase (SOD1) protein [17]. The number of known SOD1 mutations has reached more than 90 [18], but the exact mechanism leading to selective motor neuron degeneration still challenges investigators [2,5,20]. Previous reports showed that mitochondria are targeted organelles in mice carrying the G93A or G37R mutation [4,12], and that swollen and vacuolated mitochondria appear before motor neuron death, during decline in muscle strength of G93A mice [12]. In addition, dietary supplements of major energy source creatine attenuated motor neuron degeneration of G93A mice [11]. Several reports confirmed that a fraction of SOD1 is normally present in mitochondria of the central nervous system (CNS) and liver [10,14], including CNS of G93A mice [6]. Recently, in in vitro conditions Okado-Matsumoto and Fridovich [15] found a decrease of SOD1 in mitochondria specifically for * Corresponding author. Tel.: 181-86-235-7365; fax: 181-86235-7368. E-mail address: [email protected] (K. Abe).
the mutant forms of the enzyme and proposed a mechanism for the disease. We attempted to extend these observations using tissues from different ages of G93A mice and their non-transgenic littermates, and investigated for a possible change in SOD1 localization towards mitochondria by immunohistochemisty. Transgenic (Tg) mice with the G93A mutation of the human SOD1 gene (strain B6SJL-TgN (SOD1-G93A) 1 Gur dl were obtained from the Jackson Laboratory (ME, USA). Heterozygous Tg males were mated with C57B females (Shimizu Laboratory Supplies, Japan) and their progeny were identified by polymerase chain reaction of tail DNA with primers specific for exon four of human SOD1 as previously reported [20]. Non-Tg littermates, wild-type (Wt), were used as controls. All protocols were conducted in agreement with the guidelines of the Animal Committee of Graduate School of Medicine and Dentistry, Okayama University. At about 30 weeks (W) of age, G93A Tg mice developed progressive muscle weakness and spasticity in one or both hind limbs, which advanced to severe paralysis and inability to feed at 35 W. Three groups of SOD1 Tg mice at presymptomatic 8 and 20 W and symptomatic 32 W and their age-matched Wt controls were sacrificed. Spinal cords were obtained from three animals per
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 91 7- 5
54
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
Fig. 1. Double immunofluorescence labeling of SOD1 (red) and Grp75 (green) in the anterior horn of Wt (A–C) and Tg (D–F) animals at 8 (A, D), 20 (B, E) and 32 (C, F) W. Higher magnification of a typical motor neuron at each age is in inlets. Note dot-like SOD1 immunoreactivities (arrowheads) at 20 (E) and 32 (F) W in neuropil of Tg mice and a decrease of SOD1 signal in the white matter (asterisk) and motor neuron body (large arrowhead) of Tg mice at 32 W (F). Also note yellow signals in the cytoplasm of motor neurons in Wt (A–C, inlets) and presymptomatic Tg (D–E, inlets) mice, which are lost in symptomatic Tg animals at 32 W (F, inlet). Scale bars: 50 mm and 10 mm for inlets.
each group according to our previous report [20] and 10 mm sections were cut. After brief washing in phosphate-buffered saline (PBS), lumbar cord sections were blocked with 3% bovine serum albumin for 1 h at room temperature. Because a mouse primary antibody was used, additional blocking of nonspecific staining from antimouse IgG was done by applying MOM kit (PK-2200, Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instruction. Sections were incubated for 48 h at 4 8C with the two primary antibodies together in a blocking solution (1% BSA/ 0.1%Triton-X/ MOM diluent): a rabbit anti-SOD1 against human and mouse SOD1 protein (1:1000, a kind gift from Dr P.C. Wong) [1,16], and a mouse anti-glucose-regulated protein 75 (Grp75) (1:200, Cat no. SPA-825, Stressgen, Victoria, Canada). After PBS washing, sections were reacted with a mixture of secondary antibodies: biotinylated
anti-mouse IgG from the kit (1:250) and anti-rabbit rhodamine conjugated (1:500, Chemicon International, Temecula, CA, USA) for 30 min at room temperature. Grp75 immunoreactivity was visualized in three steps: after the secondary biotinylated anti-mouse antibody, sections were reacted with Fluorescein Avidin DCS (Vector Lab.) for 5 min at 4 8C and washed in PBS. Finally sections were mounted in Vectashield (H-1200, Vector Lab.) and examined with Zeiss LSM 510 confocal laser scanning microscope. Rabbit serum and normal mouse IgG1 (the same isotype as the mouse anti-Grp75 antibody) were used as controls instead of the primary antibodies giving no immunoreactivity. Both in Wt and Tg animals, large motor neurons, small interneurons and substantia gelatinosa showed similar SOD1 immunoreactivity at 8 W (Fig. 1A,D, Table 1). In Wt mice, SOD1 immunoreactivity at 8 W (Fig. 1A) did not show a change with age, and was essentially the same as ages 20 and 32 W (Fig. 1B,C). In contrast, Tg mice showed a less intensive SOD1 signal at 32 W in the cytoplasm of large motor neurons (Fig. 1F and inlet). A weak SOD1 signal was detected in the nucleus of large motor neurons at 8 W of Tg mice (Fig. 1D, inlet), which progressively became less intensive with ages at 20 and 32 W (Fig. 1E,F inlets). Again unlike Wt animals (Fig. 1A, asterisk), Tg animals revealed an intense SOD1 staining in the white matter of anterior and lateral columns at presymptomatic 8 and 20 W (Fig. 1D,E, asterisks), which was greatly reduced at symptomatic 32 W (Fig. 1F, asterisk). Furthermore, the neuropil of anterior horn showed a slight and a few dot-like SOD1 deposits at 20 W (Fig. 1E, arrowheads), which became more frequent and stronger at 32 W in the whole anterior horn (Fig. 1F, small arrowheads). Immunofluorescent analysis for Grp75 revealed tiny, green, dot-like signals in the cell bodies and processes of large motor neurons, representing mitochondria (Fig. 1). Under higher magnification, the green fluorescent intensity was greater in the cell bodies of large motor neurons than in their neuronal processes at 8 W of Wt mice (Fig. 1A, inlet). Similar pattern was found in both Wt and Tg animals with slight increase in signal intensity with age (Fig. 1, Table 2). The number of Grp75 positive dots was not reduced in the surviving large motor neurons even at 32 W of Tg mice (Fig. 1F, Table 2). As for double staining, Wt mice showed the same pattern between 8, 20 and 32 W weeks (Fig. 1A–C), and the red SOD1 and the green Grp75 signals colocalized giving yellow signals (Fig. 1A–C, inlets). The number of yellow signals was 10–12 in each large motor neuron of Wt mice. Colocalization signal in the cytoplasm was similar at 8 and 20 W of Tg mice to that of Wt mice, but was absent in symptomatic Tg mice at 32 W (Fig. 1F, inlet). In the present study, SOD1 revealed cytosolic staining in perikarya and processes of large motor neurons, small interneurons and posterior neurons as previously reported [16]. SOD1 signal in the white matter and cell nucleus (Fig. 1D–
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
55
Table 1 Immunoreactivity for SOD1 in the lumbar spinal cord of Wt and Tg mice a Age of animals (weeks)
Case number
Wt
Tg
Motor neuron
Dots in neuropil
White matter
Motor neuron
Dots in neuropil
White matter
8
1 2 3
111 111 111
2 2 2
2 2 2
111 111 111
2 2 2
11 11 11
20
1 2 3
111 111 111
2 2 2
2 2 2
111 111 111
1 1 1
11 11 11
32
1 2 3
111 111 111
2 2 2
2 2 2
1 11 1
111 111 111
^ ^ ^
a The degree of immunohistochemically reactive product was rated for semiquantitation as follows: nil (2), weak (1), moderate (1 1 ) and strong (111).
E) of presymptomatic Tg mice was also noted in other Tg mice overexpressing normal human SOD1 [1,7] and may simply be the result of SOD1 gene overexpression. In contrast, dot-like SOD1 immunoreactivities in the neuropil of anterior horn at 20 W presymptomatic (Fig. 1E, arrowheads) and 32 W symptomatic (Fig. 1F, small arrowheads) Tg animals should be a characteristic of mutant SOD1 Tg mice. Increase with age of similar dot-like SOD1 immunoreactivity in the gray matter of 12 W G93A mice was connected to aggregations of SOD1 beginning in neuronal processes [19], or could be a result of the progressive slowing of antegrade axonal transport in this model [20]. Of interest is a decrease of SOD1 signal in the white matter (Fig. 1F, asterisk), cell bodies (Fig. 1F, inlet, large arrowhead) and nucleus of motor neurons in symptomatic animals at 32 W. Decrease of SOD1 concentration in the spinal cord of symptomatic G93A animals measured by enzyme-linked immunosorbent assay and Western blotting [8] suggested a result from reduction of motor neuron number. However, Table 2 Immunoreactivity for Grp75 in the large motor neurons of lumbar spinal cord of Wt and Tg mice a Age of animals (weeks)
Case number
Wt
Tg
8
1 2 3
1 111 1
11 11 11
20
1 2 3
111 111 111
111 111 111
32
1 2 3
111 111 111
111 111 111
a The degree of immunohistochemically reactive product was rated for semiquanititation as follows: nil (2), weak (1), moderate (1 1 ) and strong (111).
the present results showed that a reduction of SOD1 protein level in each motor neuron was also present in Tg mice. Grp75 protein is a constitutively expressed member of the heat shock proteins, and resides in inner mitochondrial matrix [13]. In agreement, we found tiny dot-like immunoreactivities in the motor neuron cytoplasm (Fig. 1A–E). In both Wt and Tg animals, slight increase was noted in the green Grp75 signal with age (Fig. 1), probably due to an increasing chaperone function for protein folding in mitochondria with age [3]. However, no difference of Grp75 signals between Wt and Tg mice suggests no additional overload of mitochondrial chaperoning activity or mitochondrial stress in motor neurons of Tg animals. In the present study, Grp75 and SOD1 proteins showed colocalization in the cytoplasm of Wt and Tg animals, except for Tg animals at 32 W (Fig. 1F, inlet). SOD1 is regarded mainly as a cytosolic protein, but recent reports showed that it is also present in mitochondria of CNS and liver [6,10,14]. Data by Jaarsma et al. [8] also showed association of mutant SOD1 with vacuoles and dilated mitochondria in G93A mice. We believe that the Grp75 marker detected morphologically intact mitochondria, since Grp75 immunoreactivity did not show a change with age between Wt and Tg animals, precluding us to confirm Jaarsma’s observations. The main finding of this study is the loss of colocalization between Grp75 and SOD1 proteins in Tg symptomatic animals. This lack of colocalization could be a result of the decrease of SOD1 signal at symptomatic age (Fig. 1F). The late decrease of SOD1 signal within large motor neurons and the progressive increase of dot-like deposits in the neuropil suggest a relationship between them. SOD1 deposits in neuropil may illustrate conformational changes of SOD1 that cause impaired detection of the protein inside motor neurons. Alternatively, a detachment of SOD1 from mitochondria could be responsible for the loss of colocalization, as suggested by Kira et al., 2001 [9]. A recent report by Okado-Matsumoto and Fridovich in in vitro
56
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
conditions found a decrease of SOD1 within mitochondria specifically for the mutant forms of the protein (G93A, G37R and G41D) [15]. Thus, this study is the first in vivo observation of loss of colocalization between SOD1 and mitochondrial marker in symptomatic Tg animals. Although this late loss of colocalization may not be a triggering factor of motor neuron death, loss of SOD1 protein from mitochondria could contribute to motor neuron damage through decreased anti-oxidative defense around the organelle. The authors would like to thank Dr P.C. Wong and Dr J. Subramaniam at Johns Hopkins University, Department of Pathology for the kind gift of the rabbit anti-SOD1 antibody. This study was partly supported by a Grant-in-Aid for Scientific Research (B) 12470141 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by grants from (Tashiro K, Itoyama Y, and Tsuji S) and Comprehensive Research on Aging and Health (H11Choju-010, No.207, Koizumi A) from the Ministry of Health, Labor and Welfare of Japan. [1] Borchelt, D.R., Wong, P.C., Becher, M.W., Pardo, C.A., Lee, M.K., Xu, Z., Thinakaran, G., Jenkins, N.A., Copeland, N.G., Sisodia, S.S., Cleveland, D.W., Price, D.L. and Hoffman, P.N., Axonal transport of mutant superoxide dismutase 1 and focal axonal abnormalities in the proximal axons of transgenic mice, Neurobiol. Dis., 5 (1998) 27–35. [2] Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D., Ohama, E., Reaume, A.G., Scott, R.W. and Cleveland, D.W., Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1, Science, 281 (1998) 1851–1854. [3] Craig, E.E. and Hood, D.A., Influence of aging on protein import into cardiac mitochondria, Am. J. Physiol., 272 (1997) 2983–2988. [4] Dal Canto, M.C. and Gurney, M.E., 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 (1995) 25–40. [5] Gurney, M.E., Pu, H., Chui, A.Y., Dal Canto, M.C., Polchow, C.Y., Chen, W., Zhai, P. and Sufit, R.L., Siddique, T., Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation, Science, 264 (1994) 1772– 1775. [6] Higgins, C.M.J., Jung, C., Ding, H. and Xu, Z., Mutant CuZn superoxide dismutase that causes motor neuron degeneration is present in mitochondria in the CNS, J. Neurosci., 22 (2002) 215. [7] Jaarsma, D., Haasdijk, E.D., Grashorn, J.A.C., Hawkins, R., Duijn, W., van Verspaget, H.W., London, J. and Holstege, J.C., Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration and premature motor neuron death
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
and accelerated motor neuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1, Neurobiol. Dis., 7 (2000) 623–643. Jaarsma, D., Rognoni, F., Duijn, W., van Verspaget, H.W., Haasdijk, E.D. and Holstege, J.C., CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosislinked SOD1 mutations, Acta Neuropathol., 102 (2001) 293– 305. Kira, Y., Sato, E,, Teramura, M., Fujii, J. and Inoue, M., Subcellular localization of Cu,Zn SOD and its role in the pathogenesis of FALS, In K. Abe (Ed.), Molecular Mechanism and Therapeutics of Amyotrophic Lateral Sclerosis, Elsevier Science, Amsterdam, 2001, pp. 71–77. Kira, Y., Sato, E. and Inoue, M., Association of Cu,Zn- type superoxide dismutase with mitochondria and peroxisomes, Arch. Biochem. Biophys., 399 (2002) 96–102. Klivenyi, P., Ferrante, R.J., Matthews, R.T., Bogdanov, M.B., Klein, A.M., Andreassen, O.A., Mueller, G., Wermer, M., Kaddurah-Daouk, R. and Beal, M.F., Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis, Nat. Med., 51 (1999) 347–350. Kong, J. and Xu, Z., Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1, J. Neurosci., 18 (1998) 3241–3250. Mizzen, L.A., Chang, C., Garrels, J.I. and Welch, W.J., Identification, characterization and purification of two mammalian stress proteins present in mitochondria, Grp 75, a member of the hsp 70 family and hsp 58, homolog of the bacterial groEL protein, J. Biol. Chem., 264 (1989) 20664– 20675. Okado-Matsumoto, A. and Fridovich, I., Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Znsod in mitochondria, J. Biol. Chem., 276 (2001) 38388– 38393. Okado-Matsumoto, A. and Fridovich, I., Amyotrophic lateral sclerosis: A proposed mechanism, Proc. Natl. Acad. Sci. USA, 99 (2002) 9010–9014. Pardo, C., Xu, Z., Borchelt, D.R., Price, D.L., Sisodia, S.S. and Cleveland, D.W., Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons, Proc. Natl. Acad. Sci. USA, 92 (1995) 954–958. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Gotto, J., O’Regan, J.P. and Deng, H.X., Mutations in Cu,Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362 (1993) 59–62. Rowland, L. and Shneider, N., Amyotrophic Lateral Sclerosis, N. Engl. J. Med., 344 (2001) 1688–1700. Stieber, A., Gonatas, J. and Gonatas, N., Aggregation of ubiquitin and a mutant ALS- lined SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus, J. Neurol. Sci., 173 (2000) 53–62. Warita, H., Itoyama, Y. and Abe, K., Selective impairment of fast anterograde axonal transport in the peripheral nerves of asymptomatic transgenic mice with a G93A mutant SOD1 gene, Brain Res., 819 (1999) 120–131.
Change in superoxide dismutase1 protein localization towards mitochondria: an immunohistochemical study in transgenic G93A mice Hristelina Ilieva, Isao Nagano, Tetsuro Murakami, Mito Shiote, Yasuhiro Manabe, Koji Abe* Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 1 July 2002; received in revised form 10 August 2002; accepted 12 August 2002
Abstract Localization of superoxide dismutase1 (SOD1) and mitochondrial glucose-regulated protein 75 (Grp75), were examined in the spinal cords of transgenic (Tg) mice expressing human mutant SOD1 protein (G93A) and wild-type (Wt) controls at 8, 20 and 32 weeks. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons. Colocalization of Grp75 and SOD1 signals was demonstrated in Wt and presymptomatic Tg animals, while it was lost in Tg mice at a symptomatic age. The present results suggest that loss of SOD1 protein from mitochondria could contribute to motor neuron damage. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Immunohistochemistry; Mitochondria; Superoxide dismutase1
Amyotrophic lateral sclerosis is a fatal neurological disease characterized by selective degeneration of upper and lower motor neurons. About 5–10% of all cases are familial 20% of which are results of dominant mutations in the gene encoding a ubiquitously expressed Cu/Zn superoxide dismutase (SOD1) protein [17]. The number of known SOD1 mutations has reached more than 90 [18], but the exact mechanism leading to selective motor neuron degeneration still challenges investigators [2,5,20]. Previous reports showed that mitochondria are targeted organelles in mice carrying the G93A or G37R mutation [4,12], and that swollen and vacuolated mitochondria appear before motor neuron death, during decline in muscle strength of G93A mice [12]. In addition, dietary supplements of major energy source creatine attenuated motor neuron degeneration of G93A mice [11]. Several reports confirmed that a fraction of SOD1 is normally present in mitochondria of the central nervous system (CNS) and liver [10,14], including CNS of G93A mice [6]. Recently, in in vitro conditions Okado-Matsumoto and Fridovich [15] found a decrease of SOD1 in mitochondria specifically for * Corresponding author. Tel.: 181-86-235-7365; fax: 181-86235-7368. E-mail address: [email protected] (K. Abe).
the mutant forms of the enzyme and proposed a mechanism for the disease. We attempted to extend these observations using tissues from different ages of G93A mice and their non-transgenic littermates, and investigated for a possible change in SOD1 localization towards mitochondria by immunohistochemisty. Transgenic (Tg) mice with the G93A mutation of the human SOD1 gene (strain B6SJL-TgN (SOD1-G93A) 1 Gur dl were obtained from the Jackson Laboratory (ME, USA). Heterozygous Tg males were mated with C57B females (Shimizu Laboratory Supplies, Japan) and their progeny were identified by polymerase chain reaction of tail DNA with primers specific for exon four of human SOD1 as previously reported [20]. Non-Tg littermates, wild-type (Wt), were used as controls. All protocols were conducted in agreement with the guidelines of the Animal Committee of Graduate School of Medicine and Dentistry, Okayama University. At about 30 weeks (W) of age, G93A Tg mice developed progressive muscle weakness and spasticity in one or both hind limbs, which advanced to severe paralysis and inability to feed at 35 W. Three groups of SOD1 Tg mice at presymptomatic 8 and 20 W and symptomatic 32 W and their age-matched Wt controls were sacrificed. Spinal cords were obtained from three animals per
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 91 7- 5
54
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
Fig. 1. Double immunofluorescence labeling of SOD1 (red) and Grp75 (green) in the anterior horn of Wt (A–C) and Tg (D–F) animals at 8 (A, D), 20 (B, E) and 32 (C, F) W. Higher magnification of a typical motor neuron at each age is in inlets. Note dot-like SOD1 immunoreactivities (arrowheads) at 20 (E) and 32 (F) W in neuropil of Tg mice and a decrease of SOD1 signal in the white matter (asterisk) and motor neuron body (large arrowhead) of Tg mice at 32 W (F). Also note yellow signals in the cytoplasm of motor neurons in Wt (A–C, inlets) and presymptomatic Tg (D–E, inlets) mice, which are lost in symptomatic Tg animals at 32 W (F, inlet). Scale bars: 50 mm and 10 mm for inlets.
each group according to our previous report [20] and 10 mm sections were cut. After brief washing in phosphate-buffered saline (PBS), lumbar cord sections were blocked with 3% bovine serum albumin for 1 h at room temperature. Because a mouse primary antibody was used, additional blocking of nonspecific staining from antimouse IgG was done by applying MOM kit (PK-2200, Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instruction. Sections were incubated for 48 h at 4 8C with the two primary antibodies together in a blocking solution (1% BSA/ 0.1%Triton-X/ MOM diluent): a rabbit anti-SOD1 against human and mouse SOD1 protein (1:1000, a kind gift from Dr P.C. Wong) [1,16], and a mouse anti-glucose-regulated protein 75 (Grp75) (1:200, Cat no. SPA-825, Stressgen, Victoria, Canada). After PBS washing, sections were reacted with a mixture of secondary antibodies: biotinylated
anti-mouse IgG from the kit (1:250) and anti-rabbit rhodamine conjugated (1:500, Chemicon International, Temecula, CA, USA) for 30 min at room temperature. Grp75 immunoreactivity was visualized in three steps: after the secondary biotinylated anti-mouse antibody, sections were reacted with Fluorescein Avidin DCS (Vector Lab.) for 5 min at 4 8C and washed in PBS. Finally sections were mounted in Vectashield (H-1200, Vector Lab.) and examined with Zeiss LSM 510 confocal laser scanning microscope. Rabbit serum and normal mouse IgG1 (the same isotype as the mouse anti-Grp75 antibody) were used as controls instead of the primary antibodies giving no immunoreactivity. Both in Wt and Tg animals, large motor neurons, small interneurons and substantia gelatinosa showed similar SOD1 immunoreactivity at 8 W (Fig. 1A,D, Table 1). In Wt mice, SOD1 immunoreactivity at 8 W (Fig. 1A) did not show a change with age, and was essentially the same as ages 20 and 32 W (Fig. 1B,C). In contrast, Tg mice showed a less intensive SOD1 signal at 32 W in the cytoplasm of large motor neurons (Fig. 1F and inlet). A weak SOD1 signal was detected in the nucleus of large motor neurons at 8 W of Tg mice (Fig. 1D, inlet), which progressively became less intensive with ages at 20 and 32 W (Fig. 1E,F inlets). Again unlike Wt animals (Fig. 1A, asterisk), Tg animals revealed an intense SOD1 staining in the white matter of anterior and lateral columns at presymptomatic 8 and 20 W (Fig. 1D,E, asterisks), which was greatly reduced at symptomatic 32 W (Fig. 1F, asterisk). Furthermore, the neuropil of anterior horn showed a slight and a few dot-like SOD1 deposits at 20 W (Fig. 1E, arrowheads), which became more frequent and stronger at 32 W in the whole anterior horn (Fig. 1F, small arrowheads). Immunofluorescent analysis for Grp75 revealed tiny, green, dot-like signals in the cell bodies and processes of large motor neurons, representing mitochondria (Fig. 1). Under higher magnification, the green fluorescent intensity was greater in the cell bodies of large motor neurons than in their neuronal processes at 8 W of Wt mice (Fig. 1A, inlet). Similar pattern was found in both Wt and Tg animals with slight increase in signal intensity with age (Fig. 1, Table 2). The number of Grp75 positive dots was not reduced in the surviving large motor neurons even at 32 W of Tg mice (Fig. 1F, Table 2). As for double staining, Wt mice showed the same pattern between 8, 20 and 32 W weeks (Fig. 1A–C), and the red SOD1 and the green Grp75 signals colocalized giving yellow signals (Fig. 1A–C, inlets). The number of yellow signals was 10–12 in each large motor neuron of Wt mice. Colocalization signal in the cytoplasm was similar at 8 and 20 W of Tg mice to that of Wt mice, but was absent in symptomatic Tg mice at 32 W (Fig. 1F, inlet). In the present study, SOD1 revealed cytosolic staining in perikarya and processes of large motor neurons, small interneurons and posterior neurons as previously reported [16]. SOD1 signal in the white matter and cell nucleus (Fig. 1D–
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
55
Table 1 Immunoreactivity for SOD1 in the lumbar spinal cord of Wt and Tg mice a Age of animals (weeks)
Case number
Wt
Tg
Motor neuron
Dots in neuropil
White matter
Motor neuron
Dots in neuropil
White matter
8
1 2 3
111 111 111
2 2 2
2 2 2
111 111 111
2 2 2
11 11 11
20
1 2 3
111 111 111
2 2 2
2 2 2
111 111 111
1 1 1
11 11 11
32
1 2 3
111 111 111
2 2 2
2 2 2
1 11 1
111 111 111
^ ^ ^
a The degree of immunohistochemically reactive product was rated for semiquantitation as follows: nil (2), weak (1), moderate (1 1 ) and strong (111).
E) of presymptomatic Tg mice was also noted in other Tg mice overexpressing normal human SOD1 [1,7] and may simply be the result of SOD1 gene overexpression. In contrast, dot-like SOD1 immunoreactivities in the neuropil of anterior horn at 20 W presymptomatic (Fig. 1E, arrowheads) and 32 W symptomatic (Fig. 1F, small arrowheads) Tg animals should be a characteristic of mutant SOD1 Tg mice. Increase with age of similar dot-like SOD1 immunoreactivity in the gray matter of 12 W G93A mice was connected to aggregations of SOD1 beginning in neuronal processes [19], or could be a result of the progressive slowing of antegrade axonal transport in this model [20]. Of interest is a decrease of SOD1 signal in the white matter (Fig. 1F, asterisk), cell bodies (Fig. 1F, inlet, large arrowhead) and nucleus of motor neurons in symptomatic animals at 32 W. Decrease of SOD1 concentration in the spinal cord of symptomatic G93A animals measured by enzyme-linked immunosorbent assay and Western blotting [8] suggested a result from reduction of motor neuron number. However, Table 2 Immunoreactivity for Grp75 in the large motor neurons of lumbar spinal cord of Wt and Tg mice a Age of animals (weeks)
Case number
Wt
Tg
8
1 2 3
1 111 1
11 11 11
20
1 2 3
111 111 111
111 111 111
32
1 2 3
111 111 111
111 111 111
a The degree of immunohistochemically reactive product was rated for semiquanititation as follows: nil (2), weak (1), moderate (1 1 ) and strong (111).
the present results showed that a reduction of SOD1 protein level in each motor neuron was also present in Tg mice. Grp75 protein is a constitutively expressed member of the heat shock proteins, and resides in inner mitochondrial matrix [13]. In agreement, we found tiny dot-like immunoreactivities in the motor neuron cytoplasm (Fig. 1A–E). In both Wt and Tg animals, slight increase was noted in the green Grp75 signal with age (Fig. 1), probably due to an increasing chaperone function for protein folding in mitochondria with age [3]. However, no difference of Grp75 signals between Wt and Tg mice suggests no additional overload of mitochondrial chaperoning activity or mitochondrial stress in motor neurons of Tg animals. In the present study, Grp75 and SOD1 proteins showed colocalization in the cytoplasm of Wt and Tg animals, except for Tg animals at 32 W (Fig. 1F, inlet). SOD1 is regarded mainly as a cytosolic protein, but recent reports showed that it is also present in mitochondria of CNS and liver [6,10,14]. Data by Jaarsma et al. [8] also showed association of mutant SOD1 with vacuoles and dilated mitochondria in G93A mice. We believe that the Grp75 marker detected morphologically intact mitochondria, since Grp75 immunoreactivity did not show a change with age between Wt and Tg animals, precluding us to confirm Jaarsma’s observations. The main finding of this study is the loss of colocalization between Grp75 and SOD1 proteins in Tg symptomatic animals. This lack of colocalization could be a result of the decrease of SOD1 signal at symptomatic age (Fig. 1F). The late decrease of SOD1 signal within large motor neurons and the progressive increase of dot-like deposits in the neuropil suggest a relationship between them. SOD1 deposits in neuropil may illustrate conformational changes of SOD1 that cause impaired detection of the protein inside motor neurons. Alternatively, a detachment of SOD1 from mitochondria could be responsible for the loss of colocalization, as suggested by Kira et al., 2001 [9]. A recent report by Okado-Matsumoto and Fridovich in in vitro
56
H. Ilieva et al. / Neuroscience Letters 332 (2002) 53–56
conditions found a decrease of SOD1 within mitochondria specifically for the mutant forms of the protein (G93A, G37R and G41D) [15]. Thus, this study is the first in vivo observation of loss of colocalization between SOD1 and mitochondrial marker in symptomatic Tg animals. Although this late loss of colocalization may not be a triggering factor of motor neuron death, loss of SOD1 protein from mitochondria could contribute to motor neuron damage through decreased anti-oxidative defense around the organelle. The authors would like to thank Dr P.C. Wong and Dr J. Subramaniam at Johns Hopkins University, Department of Pathology for the kind gift of the rabbit anti-SOD1 antibody. This study was partly supported by a Grant-in-Aid for Scientific Research (B) 12470141 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by grants from (Tashiro K, Itoyama Y, and Tsuji S) and Comprehensive Research on Aging and Health (H11Choju-010, No.207, Koizumi A) from the Ministry of Health, Labor and Welfare of Japan. [1] Borchelt, D.R., Wong, P.C., Becher, M.W., Pardo, C.A., Lee, M.K., Xu, Z., Thinakaran, G., Jenkins, N.A., Copeland, N.G., Sisodia, S.S., Cleveland, D.W., Price, D.L. and Hoffman, P.N., Axonal transport of mutant superoxide dismutase 1 and focal axonal abnormalities in the proximal axons of transgenic mice, Neurobiol. Dis., 5 (1998) 27–35. [2] Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D., Ohama, E., Reaume, A.G., Scott, R.W. and Cleveland, D.W., Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1, Science, 281 (1998) 1851–1854. [3] Craig, E.E. and Hood, D.A., Influence of aging on protein import into cardiac mitochondria, Am. J. Physiol., 272 (1997) 2983–2988. [4] Dal Canto, M.C. and Gurney, M.E., 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 (1995) 25–40. [5] Gurney, M.E., Pu, H., Chui, A.Y., Dal Canto, M.C., Polchow, C.Y., Chen, W., Zhai, P. and Sufit, R.L., Siddique, T., Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation, Science, 264 (1994) 1772– 1775. [6] Higgins, C.M.J., Jung, C., Ding, H. and Xu, Z., Mutant CuZn superoxide dismutase that causes motor neuron degeneration is present in mitochondria in the CNS, J. Neurosci., 22 (2002) 215. [7] Jaarsma, D., Haasdijk, E.D., Grashorn, J.A.C., Hawkins, R., Duijn, W., van Verspaget, H.W., London, J. and Holstege, J.C., Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration and premature motor neuron death
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
and accelerated motor neuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1, Neurobiol. Dis., 7 (2000) 623–643. Jaarsma, D., Rognoni, F., Duijn, W., van Verspaget, H.W., Haasdijk, E.D. and Holstege, J.C., CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosislinked SOD1 mutations, Acta Neuropathol., 102 (2001) 293– 305. Kira, Y., Sato, E,, Teramura, M., Fujii, J. and Inoue, M., Subcellular localization of Cu,Zn SOD and its role in the pathogenesis of FALS, In K. Abe (Ed.), Molecular Mechanism and Therapeutics of Amyotrophic Lateral Sclerosis, Elsevier Science, Amsterdam, 2001, pp. 71–77. Kira, Y., Sato, E. and Inoue, M., Association of Cu,Zn- type superoxide dismutase with mitochondria and peroxisomes, Arch. Biochem. Biophys., 399 (2002) 96–102. Klivenyi, P., Ferrante, R.J., Matthews, R.T., Bogdanov, M.B., Klein, A.M., Andreassen, O.A., Mueller, G., Wermer, M., Kaddurah-Daouk, R. and Beal, M.F., Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis, Nat. Med., 51 (1999) 347–350. Kong, J. and Xu, Z., Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1, J. Neurosci., 18 (1998) 3241–3250. Mizzen, L.A., Chang, C., Garrels, J.I. and Welch, W.J., Identification, characterization and purification of two mammalian stress proteins present in mitochondria, Grp 75, a member of the hsp 70 family and hsp 58, homolog of the bacterial groEL protein, J. Biol. Chem., 264 (1989) 20664– 20675. Okado-Matsumoto, A. and Fridovich, I., Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Znsod in mitochondria, J. Biol. Chem., 276 (2001) 38388– 38393. Okado-Matsumoto, A. and Fridovich, I., Amyotrophic lateral sclerosis: A proposed mechanism, Proc. Natl. Acad. Sci. USA, 99 (2002) 9010–9014. Pardo, C., Xu, Z., Borchelt, D.R., Price, D.L., Sisodia, S.S. and Cleveland, D.W., Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons, Proc. Natl. Acad. Sci. USA, 92 (1995) 954–958. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Gotto, J., O’Regan, J.P. and Deng, H.X., Mutations in Cu,Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362 (1993) 59–62. Rowland, L. and Shneider, N., Amyotrophic Lateral Sclerosis, N. Engl. J. Med., 344 (2001) 1688–1700. Stieber, A., Gonatas, J. and Gonatas, N., Aggregation of ubiquitin and a mutant ALS- lined SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus, J. Neurol. Sci., 173 (2000) 53–62. Warita, H., Itoyama, Y. and Abe, K., Selective impairment of fast anterograde axonal transport in the peripheral nerves of asymptomatic transgenic mice with a G93A mutant SOD1 gene, Brain Res., 819 (1999) 120–131.