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Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene
Neuroscience Letters 308 (2001) 149±152
www.elsevier.com/locate/neulet
Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene T. Murakami a, I. Nagano a, T. Hayashi a, Y. Manabe a, M. Shoji a, Y. Setoguchi b, K. Abe a,* a
Department of Neurology, Okayama University, Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, Japan b Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan Received 8 March 2001; received in revised form 30 May 2001; accepted 31 May 2001
Abstract A replication-defective recombinant adenoviral vector containing E. coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age. After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice. Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively. These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: lacZ gene; Amyotrophic lateral sclerosis; Superoxide dismutase; Transgenic mice; Retrograde axonal transport
Amyotrophic lateral sclerosis (ALS) is a disease causing progressive weakness in skeletal muscles due to loss of motor neurons in the spinal cord. Around 5±10% of ALS are familial (FALS), and over 60 types of mutations have been reported in the gene coding Cu/Zn superoxide dismutase (superoxide dismutase 1, SOD1) in about 20% of FALS cases [3,10]. Transgenic mice carrying a missense mutation in the human SOD1 gene, which show the similar phenotype to those of the human patients, have been used as an animal model for ALS. Recent studies using those transgenic mice show that anterograde axonal transport in the peripheral nerve is impaired in the mice [14,15]. In this study, we injected replication-defective recombinant adenoviral vector containing E. coli lacZ gene into the muscles of transgenic (Tg) and non-transgenic wild type (WT) mice, and examined how lacZ was expressed in the muscles, peripheral nerves, and the spinal cords. Transgenic mice expressing mutant human SOD1 gene containing an amino acid substitution of glycine by alanine at codon 93 (G93A) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Symptomatic G93A transgenic mice at the end stage of the disease (40 weeks old, n 4) * Corresponding author. Tel.: 181-86-2357365; fax: 181-862357368. E-mail address: [email protected] (K. Abe).
and WT littermates (n 4) were examined in this study. According to our previous reports [1,2], the recombinant adenoviral vector (Ad-CMV-lacZ) used in this study expresses the E. coli lacZ gene under the control of the human cytomegalovirus (CMV) promoter with the nuclear localizing signal of simian virus 40 (SV40) [6]. Under anesthesia with inhalation of a nitrous-oxide/ oxygen/ iso¯urane (68:30:2) mixture, Ad-CMV-lacZ (2.5 £ 10 9 plaque forming unit in 5 ml of vector vehicle consisting of 10 mM Tris±HCl (pH 7.4), 1 mM MgCl2, and 10% glycerol) was injected into the center of bilateral gastrocnemius muscles of the mice with 10 ml Hamilton syringe (the Gastight Highperformance Syringe; Hamilton, Reno, NV, USA). To evaluate the gene transfer and expression, the animals were decapitated at 30, 60 and 90 h after the injection. Lumbar cords and full length of sciatic nerves were obtained and frozen in the powdered dry ice, and the gastrocnemius muscles were also frozen quickly in 2-methylbutane chilled by liquid nitrogen. Sciatic nerve was cut at the distal part where it enters the gastrocnemius muscle, and at the proximal end where it enters the spinal cord. Sections (10 mm thickness) of the gastrocnemius muscles, sciatic nerves, and lumbar cords were prepared on a cryostat at 2208C and stained with X-Gal (5-Bromo-4-chloro-3-indolyl-beta-dgalactopyranoside; Boehringer Mannheim Biochemica,
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 03 6- 5
150
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
Fig. 1. Microphotographs of the gastrocnemius muscles (a, c, e and g) and sciatic nerves (b, d, f, and h) in WT (a, b, e and f) and Tg (c, d, g and h) mice at 60 and 90 h of injection. Compared to WT (a, e), there was obvious group atrophy in the muscles of Tg (c, g). However, no difference in the number of the nuclei stained with X-Gal (a, c and e, g). Distal sides of the sciatic nerves are located on the left of the photographs (b, d, f and h). Compared to WT (b, f), the number of the dots (arrows) and the distance is greatly reduced in Tg (d, h).
Mannheim, Germany) according to our previous reports [1,2,13]. After 4 h of reaction with X-Gal at 378C, the sections were counterstained with hematoxylin. In gastrocnemius muscles, the number of the nuclei which were stained blue was counted on the light microscope with transverse sections within 1 mm distance from the injected point. In sciatic nerves, the number of blue color-stained `dots' and the distances from the distal end of the nerve to the dots were measured with longitudinal sections. Transverse sections of lumbar cords that project axons to gastrocnemius muscle through sciatic nerve were also examined to observe the expression of lacZ protein. Data of both sides were averaged, and statistical analysis was performed with Student t-test (n 4). As compared to WT mice (Fig. 1a,e), G93A Tg mice
showed group atrophy in the gastrocnemius muscle (Fig. 1c,g). However, the numbers of the nuclei which were stained with X-Gal were 27.9 ^ 6.3 and 26.6 ^ 5.2, and 72.9 ^ 6.3 and 68.6 ^ 9.8 (mean ^ SD, /mm 2) at 60 and 90 h, respectively, and there was no difference between Tg and WT mice in all sections at 30, 60, and 90 h after the injection (Table 1, P 0:87). In sciatic nerves, no positive staining of lacZ was observed at 30 h of injection both in WT and Tg mice. However, at 60 h, great differences in the number and the distances from the distal end were found between WT and Tg mice (Fig. 1b,d,f,h). At 60 h, the numbers of lacZ positive dots were 51.0 ^ 9.3 and 3.0 ^ 0.9 (mean ^ SD/nerve slice), and the distances from the distal end were 1125 ^ 553 and 380 ^ 182 (mean ^ SD, mm), in WT and Tg mice, respectively. At 90 h, the
Table 1 The number and distance of Lac-Z staining in gastrocnemius muscles and sciatic nerves a
WT Tg a
Gastrocnemius muscles
Sciatic nerve
N
N
Distance
60 h
90 h
60 h
90 h
60 h
90 h
27.9 ^ 6.3 26.6 ^ 5.2
72.9 ^ 8.2 68.6 ^ 9.8
51.0 ^ 9.3 3.0 ^ 0.9*
129.0 ^ 28.7 4.1 ^ 2.1*
1125 ^ 553 380 ^ 182*
3639 ^ 1175 443 ^ 201*
Data are expressed as mean ^ SD (n 4 of averaged bilateral sides). Number in gastrocnemius muscles (/mm 2). Number (/nerve slice) and distance (mm from the muscle end) in sciatic nerve. *P , 0:001 against WT group.
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
numbers of dots were 129.0 ^ 28.7 and 4.1 ^ 2.1 (mean ^ SD/nerve slice) and the distances were 3639 ^ 1175 and 443 ^ 201 (mean ^ SD, mm) in WT and Tg, respectively (Table 1). Lumbar cords were not positive for lacZ both in WT and Tg mice until 90 h of the study (not shown). The axonal transport system is divided into three classes: slow component a (SCa), slow component b (SCb), and fast component (FC) based on the transport velocity and transported proteins. SCa consists of neuro®lament, and SCb of micro®laments, calmodulin, heat shock proteins and anexins. FC consists of organelles, microtubules, and their associated proteins. SCa, SCb, and FC carry proteins at 0.2±1, 2±8, and 50±400 mm/day, respectively. Neuro®laments, which accumulate in the axon hillock of spinal motor neurons in ALS patients and the Tg mice, are transported anterogradely by SCa. In SOD1 Tg mice, several reports have suggested that both fast and slow components are impaired from the early stage of the disease [4,5,11,12,14,15]. Our previous report showed that the same adenovirus infected and expressed lacZ in the muscles of the Tg mice [13], and suggested that LacZ expression in the spinal motor neuron which was found at 7 days after the injection into the muscle was mainly attributed to the delivery of the virus vector by retrograde axonal transport. However, in the present study, there was no staining of lacZ in the lumbar cords of the mice until 90 h after the injection. On the other had, lacZ positive dots were found in the distal part of sciatic nerve (muscle side). Thus, all lacZ staining in the sciatic nerve (Fig. 1b,d,f,h) derived from proteins which were expressed in the muscles and retrogradely transported, but not from motor neurons in the anterior horn carried by anterograde axonal transport. For the delivery of lacZ protein, adenovirus carrying lacZ gene has to infect the muscle, then the gene has to be expressed in the muscle. As shown in Fig. 1a,c,e,g and Table 1, the numbers of lacZ positive nuclei were the same between WT and Tg mice groups. Therefore, the delay of the delivery within sciatic nerve in Tg mice (Table 1) should be attributable to the delay of the retrograde transport in Tg mice. With the distances of 60 and 90 h, mean velocities of retrograde transport for lacZ may be estimated to be 2.1 and 0.05 mm/24 h in WT and Tg, respectively. By comparing the staining of cytoplasmic dynein at both ends of the ligated sciatic nerve between WT and Tg mice, we previously reported that the fast retrograde axonal transport was impaired in young asymptomatic Tg mice [14]. With another animal model for motor neuron disease, Mitsumoto et al. showed that retrograde axonal transport was also impaired in wobbler mice, although only the amount of retrograde transport for horseradish peroxidase protein was reduced without reduction of the velocity [7,8]. In contrast, in the Tg mice, both the number and the velocity of retrograde transport were greatly decreased (Table 1, Fig. 1). The decrease in the amount of retrograde transport is probably due to the loss of motor neurons for the sciatic nerve [7,9,14].
151
In this report, we ®rst showed that the velocity and amount of retrograde axonal transport is impaired in the mice carrying mutant SOD1 gene. Because three such classes of axonal transport (SCa, SCb, and FC) were originally classi®ed for anterograde transport, the classi®cation may not be directly applied to retrograde transport. However, the present results suggest a relatively slow speed of retrograde transport for lacZ protein corresponding to the slow components in anterograde transport in WT mice, and a great reduction of the speed in Tg mice at the end stage. Impaired retrograde axonal transport of foreign gene by viral vector may provide a future potential to reveal the pathogenesis and even to evaluate axonal ¯ow in ALS patients. This work was partly supported by Grant-in-Aid for Scienti®c Research (B) 12470141 from the Ministry of Education, Science, Culture and Sports of Japan, and by grants (K.T., Y.I, S.T.) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, A.K.) from the Ministry of Health and Welfare of Japan.
[1] Abe, K., Setoguchi, Y., Hayashi, T. and Itoyama, Y., Dissociative expression of adenoviral mediated E. Coli lacZ gene between ischemic and reperfused brains, Neurosci. Lett., 226 (1997) 53±56. [2] Abe, K., Setoguchi, Y., Hayashi, T. and Itoyama, Y., In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused brain, Brain Res., 763 (1997) 191± 201. [3] Aoki, M., Abe, K., Houi, K., Ogasawara, M., Matsubara, Y., Kobayashi, T., Mochio, S., Narisawa, K. and Itoyama, Y., Variance of age at onset in a Japanese family with amyotrophic lateral sclerosis associated with a novel Cu/Zn superoxide dismutase mutation, Ann. Neurol., 37 (1995) 676±679. [4] Borchelt, D.R., Wong, P.C., Becher, M.W., Pardo, C.A., Lee, M.K., Xu, Z.S., 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., 1 (1998) 27±35. [5] Collard, J.F., Cote, F. and Julien, J.P., Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis, Nature, 375 (1995) 61±64. [6] Ghadge, G.D., Roos, R.P., Kang, U.J., Wollmann, R., Fishman, P.S., Kalynych, A.M., Barr, E. and Leiden, J.M., CNS gene delivery by retrograde transport of recombinant replication-defective adenovirus, Gene Ther., 2 (1995) 132±137. [7] Mitsumoto, H., Ferut, A.L., Kurahashi, K. and McQuarrie, I.G., Impairment of retrograde axonal transport in wobbler mouse motor neuron disease, Muscle Nerve, 13 (1990) 121± 126. [8] Mitsumoto, H., Kurahashi, K., Jacob, J.M. and McQuarrie, I.G., Retardation of fast axonal transport in wobbler mice, Muscle Nerve, 16 (1993) 542±547. [9] Mohajeri, M.H., Figlewicz, D.A. and Bohn, M.C., Selective loss of alpha motoneurons innervating the medial gastrocnemius muscle in a mouse model of amyotrophic lateral sclerosis, Exp. Neurol., 150 (1998) 329±336. [10] Orrell, R.W., Amyotrophic lateral sclerosis: copper/zinc
152
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
superoxide dismutase (SOD1) gene mutations, Neuromuscular Disord., 10 (2000) 63±68. [11] Sasaki, S. and Iwata, M., Impairment of fast axonal transport in the proximal axons of anterior horn neurons in amyotrophic lateral sclerosis, Neurology, 47 (1996) 535± 540. [12] Toyoshima, I., Sugawara, M., Kato, K., Wada, C., Hirota, K., Hasegawa, K., Kowa, H., Sheetz, M.P. and Masamune, O., Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease, J. Neurol. Sci., 159 (1998) 38±44. [13] Warita, H., Abe, K., Setoguchi, Y. and Itoyama, Y., Expression of adenovirus-mediated E. coli lacZ gene in skeletal
muscles and spinal motor neurons of transgenic mice with a mutant superoxide dismutase gene, Neurosci. Lett., 246 (1998) 153±156. [14] 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. [15] Williamson, T.L. and Cleveland, D.W., Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons, Nature Neurosci., 1 (1999) 50±56.
www.elsevier.com/locate/neulet
Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene T. Murakami a, I. Nagano a, T. Hayashi a, Y. Manabe a, M. Shoji a, Y. Setoguchi b, K. Abe a,* a
Department of Neurology, Okayama University, Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, Japan b Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan Received 8 March 2001; received in revised form 30 May 2001; accepted 31 May 2001
Abstract A replication-defective recombinant adenoviral vector containing E. coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age. After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice. Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively. These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: lacZ gene; Amyotrophic lateral sclerosis; Superoxide dismutase; Transgenic mice; Retrograde axonal transport
Amyotrophic lateral sclerosis (ALS) is a disease causing progressive weakness in skeletal muscles due to loss of motor neurons in the spinal cord. Around 5±10% of ALS are familial (FALS), and over 60 types of mutations have been reported in the gene coding Cu/Zn superoxide dismutase (superoxide dismutase 1, SOD1) in about 20% of FALS cases [3,10]. Transgenic mice carrying a missense mutation in the human SOD1 gene, which show the similar phenotype to those of the human patients, have been used as an animal model for ALS. Recent studies using those transgenic mice show that anterograde axonal transport in the peripheral nerve is impaired in the mice [14,15]. In this study, we injected replication-defective recombinant adenoviral vector containing E. coli lacZ gene into the muscles of transgenic (Tg) and non-transgenic wild type (WT) mice, and examined how lacZ was expressed in the muscles, peripheral nerves, and the spinal cords. Transgenic mice expressing mutant human SOD1 gene containing an amino acid substitution of glycine by alanine at codon 93 (G93A) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Symptomatic G93A transgenic mice at the end stage of the disease (40 weeks old, n 4) * Corresponding author. Tel.: 181-86-2357365; fax: 181-862357368. E-mail address: [email protected] (K. Abe).
and WT littermates (n 4) were examined in this study. According to our previous reports [1,2], the recombinant adenoviral vector (Ad-CMV-lacZ) used in this study expresses the E. coli lacZ gene under the control of the human cytomegalovirus (CMV) promoter with the nuclear localizing signal of simian virus 40 (SV40) [6]. Under anesthesia with inhalation of a nitrous-oxide/ oxygen/ iso¯urane (68:30:2) mixture, Ad-CMV-lacZ (2.5 £ 10 9 plaque forming unit in 5 ml of vector vehicle consisting of 10 mM Tris±HCl (pH 7.4), 1 mM MgCl2, and 10% glycerol) was injected into the center of bilateral gastrocnemius muscles of the mice with 10 ml Hamilton syringe (the Gastight Highperformance Syringe; Hamilton, Reno, NV, USA). To evaluate the gene transfer and expression, the animals were decapitated at 30, 60 and 90 h after the injection. Lumbar cords and full length of sciatic nerves were obtained and frozen in the powdered dry ice, and the gastrocnemius muscles were also frozen quickly in 2-methylbutane chilled by liquid nitrogen. Sciatic nerve was cut at the distal part where it enters the gastrocnemius muscle, and at the proximal end where it enters the spinal cord. Sections (10 mm thickness) of the gastrocnemius muscles, sciatic nerves, and lumbar cords were prepared on a cryostat at 2208C and stained with X-Gal (5-Bromo-4-chloro-3-indolyl-beta-dgalactopyranoside; Boehringer Mannheim Biochemica,
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 03 6- 5
150
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
Fig. 1. Microphotographs of the gastrocnemius muscles (a, c, e and g) and sciatic nerves (b, d, f, and h) in WT (a, b, e and f) and Tg (c, d, g and h) mice at 60 and 90 h of injection. Compared to WT (a, e), there was obvious group atrophy in the muscles of Tg (c, g). However, no difference in the number of the nuclei stained with X-Gal (a, c and e, g). Distal sides of the sciatic nerves are located on the left of the photographs (b, d, f and h). Compared to WT (b, f), the number of the dots (arrows) and the distance is greatly reduced in Tg (d, h).
Mannheim, Germany) according to our previous reports [1,2,13]. After 4 h of reaction with X-Gal at 378C, the sections were counterstained with hematoxylin. In gastrocnemius muscles, the number of the nuclei which were stained blue was counted on the light microscope with transverse sections within 1 mm distance from the injected point. In sciatic nerves, the number of blue color-stained `dots' and the distances from the distal end of the nerve to the dots were measured with longitudinal sections. Transverse sections of lumbar cords that project axons to gastrocnemius muscle through sciatic nerve were also examined to observe the expression of lacZ protein. Data of both sides were averaged, and statistical analysis was performed with Student t-test (n 4). As compared to WT mice (Fig. 1a,e), G93A Tg mice
showed group atrophy in the gastrocnemius muscle (Fig. 1c,g). However, the numbers of the nuclei which were stained with X-Gal were 27.9 ^ 6.3 and 26.6 ^ 5.2, and 72.9 ^ 6.3 and 68.6 ^ 9.8 (mean ^ SD, /mm 2) at 60 and 90 h, respectively, and there was no difference between Tg and WT mice in all sections at 30, 60, and 90 h after the injection (Table 1, P 0:87). In sciatic nerves, no positive staining of lacZ was observed at 30 h of injection both in WT and Tg mice. However, at 60 h, great differences in the number and the distances from the distal end were found between WT and Tg mice (Fig. 1b,d,f,h). At 60 h, the numbers of lacZ positive dots were 51.0 ^ 9.3 and 3.0 ^ 0.9 (mean ^ SD/nerve slice), and the distances from the distal end were 1125 ^ 553 and 380 ^ 182 (mean ^ SD, mm), in WT and Tg mice, respectively. At 90 h, the
Table 1 The number and distance of Lac-Z staining in gastrocnemius muscles and sciatic nerves a
WT Tg a
Gastrocnemius muscles
Sciatic nerve
N
N
Distance
60 h
90 h
60 h
90 h
60 h
90 h
27.9 ^ 6.3 26.6 ^ 5.2
72.9 ^ 8.2 68.6 ^ 9.8
51.0 ^ 9.3 3.0 ^ 0.9*
129.0 ^ 28.7 4.1 ^ 2.1*
1125 ^ 553 380 ^ 182*
3639 ^ 1175 443 ^ 201*
Data are expressed as mean ^ SD (n 4 of averaged bilateral sides). Number in gastrocnemius muscles (/mm 2). Number (/nerve slice) and distance (mm from the muscle end) in sciatic nerve. *P , 0:001 against WT group.
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
numbers of dots were 129.0 ^ 28.7 and 4.1 ^ 2.1 (mean ^ SD/nerve slice) and the distances were 3639 ^ 1175 and 443 ^ 201 (mean ^ SD, mm) in WT and Tg, respectively (Table 1). Lumbar cords were not positive for lacZ both in WT and Tg mice until 90 h of the study (not shown). The axonal transport system is divided into three classes: slow component a (SCa), slow component b (SCb), and fast component (FC) based on the transport velocity and transported proteins. SCa consists of neuro®lament, and SCb of micro®laments, calmodulin, heat shock proteins and anexins. FC consists of organelles, microtubules, and their associated proteins. SCa, SCb, and FC carry proteins at 0.2±1, 2±8, and 50±400 mm/day, respectively. Neuro®laments, which accumulate in the axon hillock of spinal motor neurons in ALS patients and the Tg mice, are transported anterogradely by SCa. In SOD1 Tg mice, several reports have suggested that both fast and slow components are impaired from the early stage of the disease [4,5,11,12,14,15]. Our previous report showed that the same adenovirus infected and expressed lacZ in the muscles of the Tg mice [13], and suggested that LacZ expression in the spinal motor neuron which was found at 7 days after the injection into the muscle was mainly attributed to the delivery of the virus vector by retrograde axonal transport. However, in the present study, there was no staining of lacZ in the lumbar cords of the mice until 90 h after the injection. On the other had, lacZ positive dots were found in the distal part of sciatic nerve (muscle side). Thus, all lacZ staining in the sciatic nerve (Fig. 1b,d,f,h) derived from proteins which were expressed in the muscles and retrogradely transported, but not from motor neurons in the anterior horn carried by anterograde axonal transport. For the delivery of lacZ protein, adenovirus carrying lacZ gene has to infect the muscle, then the gene has to be expressed in the muscle. As shown in Fig. 1a,c,e,g and Table 1, the numbers of lacZ positive nuclei were the same between WT and Tg mice groups. Therefore, the delay of the delivery within sciatic nerve in Tg mice (Table 1) should be attributable to the delay of the retrograde transport in Tg mice. With the distances of 60 and 90 h, mean velocities of retrograde transport for lacZ may be estimated to be 2.1 and 0.05 mm/24 h in WT and Tg, respectively. By comparing the staining of cytoplasmic dynein at both ends of the ligated sciatic nerve between WT and Tg mice, we previously reported that the fast retrograde axonal transport was impaired in young asymptomatic Tg mice [14]. With another animal model for motor neuron disease, Mitsumoto et al. showed that retrograde axonal transport was also impaired in wobbler mice, although only the amount of retrograde transport for horseradish peroxidase protein was reduced without reduction of the velocity [7,8]. In contrast, in the Tg mice, both the number and the velocity of retrograde transport were greatly decreased (Table 1, Fig. 1). The decrease in the amount of retrograde transport is probably due to the loss of motor neurons for the sciatic nerve [7,9,14].
151
In this report, we ®rst showed that the velocity and amount of retrograde axonal transport is impaired in the mice carrying mutant SOD1 gene. Because three such classes of axonal transport (SCa, SCb, and FC) were originally classi®ed for anterograde transport, the classi®cation may not be directly applied to retrograde transport. However, the present results suggest a relatively slow speed of retrograde transport for lacZ protein corresponding to the slow components in anterograde transport in WT mice, and a great reduction of the speed in Tg mice at the end stage. Impaired retrograde axonal transport of foreign gene by viral vector may provide a future potential to reveal the pathogenesis and even to evaluate axonal ¯ow in ALS patients. This work was partly supported by Grant-in-Aid for Scienti®c Research (B) 12470141 from the Ministry of Education, Science, Culture and Sports of Japan, and by grants (K.T., Y.I, S.T.) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, A.K.) from the Ministry of Health and Welfare of Japan.
[1] Abe, K., Setoguchi, Y., Hayashi, T. and Itoyama, Y., Dissociative expression of adenoviral mediated E. Coli lacZ gene between ischemic and reperfused brains, Neurosci. Lett., 226 (1997) 53±56. [2] Abe, K., Setoguchi, Y., Hayashi, T. and Itoyama, Y., In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused brain, Brain Res., 763 (1997) 191± 201. [3] Aoki, M., Abe, K., Houi, K., Ogasawara, M., Matsubara, Y., Kobayashi, T., Mochio, S., Narisawa, K. and Itoyama, Y., Variance of age at onset in a Japanese family with amyotrophic lateral sclerosis associated with a novel Cu/Zn superoxide dismutase mutation, Ann. Neurol., 37 (1995) 676±679. [4] Borchelt, D.R., Wong, P.C., Becher, M.W., Pardo, C.A., Lee, M.K., Xu, Z.S., 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., 1 (1998) 27±35. [5] Collard, J.F., Cote, F. and Julien, J.P., Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis, Nature, 375 (1995) 61±64. [6] Ghadge, G.D., Roos, R.P., Kang, U.J., Wollmann, R., Fishman, P.S., Kalynych, A.M., Barr, E. and Leiden, J.M., CNS gene delivery by retrograde transport of recombinant replication-defective adenovirus, Gene Ther., 2 (1995) 132±137. [7] Mitsumoto, H., Ferut, A.L., Kurahashi, K. and McQuarrie, I.G., Impairment of retrograde axonal transport in wobbler mouse motor neuron disease, Muscle Nerve, 13 (1990) 121± 126. [8] Mitsumoto, H., Kurahashi, K., Jacob, J.M. and McQuarrie, I.G., Retardation of fast axonal transport in wobbler mice, Muscle Nerve, 16 (1993) 542±547. [9] Mohajeri, M.H., Figlewicz, D.A. and Bohn, M.C., Selective loss of alpha motoneurons innervating the medial gastrocnemius muscle in a mouse model of amyotrophic lateral sclerosis, Exp. Neurol., 150 (1998) 329±336. [10] Orrell, R.W., Amyotrophic lateral sclerosis: copper/zinc
152
T. Murakami et al. / Neuroscience Letters 308 (2001) 149±152
superoxide dismutase (SOD1) gene mutations, Neuromuscular Disord., 10 (2000) 63±68. [11] Sasaki, S. and Iwata, M., Impairment of fast axonal transport in the proximal axons of anterior horn neurons in amyotrophic lateral sclerosis, Neurology, 47 (1996) 535± 540. [12] Toyoshima, I., Sugawara, M., Kato, K., Wada, C., Hirota, K., Hasegawa, K., Kowa, H., Sheetz, M.P. and Masamune, O., Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease, J. Neurol. Sci., 159 (1998) 38±44. [13] Warita, H., Abe, K., Setoguchi, Y. and Itoyama, Y., Expression of adenovirus-mediated E. coli lacZ gene in skeletal
muscles and spinal motor neurons of transgenic mice with a mutant superoxide dismutase gene, Neurosci. Lett., 246 (1998) 153±156. [14] 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. [15] Williamson, T.L. and Cleveland, D.W., Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons, Nature Neurosci., 1 (1999) 50±56.