Bram Research, 582 (1992) 342-345 © 1992 Elsevier Science Pubhshers B V All rights reserved. 0006-8993/92/$05 00

BRES 25211

Differential regulation of manganese and copper/zinc superoxide dismutases by the facial nerve transection T. Yoneda, S. Inagaki, Y. Hayashi, T. Nomura and H. Takagi Ftrst Department of Anatomy, Osaka Ctty Umverstty Medtcal School, Osaka (Japan)

(Accepted 25 February 1992) Key words Superoxlde &smutase, Motoneuron, Axotomy, Facial nerve, In sltu hybn&zatlon

The effects of umlateral nerve transection on manganese and copper/zinc superoxlde dlsmutase (Mn-SOD and Cu/Zn-SOD) mRNA levels m the facial nucleus were stu&ed by m sltu hybridization. An increase of Mn-SOD mRNA levels was first seen m the lpsllateral faoal nucleus 12 h after axotomy, and was most pronounced at 4-7 days after this procedure, by 56 days, the increase disappeared. There was no change in Cu/Zn-SOD mRNA levels at any time after axotomy We further confirmed, by lmmunohistochemlstry, that the increase m MnSOD transcription was followed by protein synthesis. These results are suggestwe of an important role for Mn-SOD in defense, regenerauon and recovery responses following nerve transection. It is generally accepted that cellular oxygen toxicity occurs following the oxygen-dependent generation of free radicals 4. Cells are protected from free radicals by a chemical and enzymatic antioxidant defense system, the first enzymes involved in this system being superoxide dismutases (SODs), metalloproteins which are found in both procaryotic and eucaryotic cells 2. Eucaryotic cells contain three of these enzymes; one, manganese SOD (Mn-SOD), is found in the mitochondrial matrix, another, copper/zinc SOD (Cu/Zn-SOD), is found throughout the cell, and the third one is an extracellular SOD similar to the Cu/Zn enzyme 1"3. Mn-SOD induction may be important for cell defense against such oxidative stresses as inflammation, immunoresponse and the action of redox-active compounds, since induction of MnSOD synthesis has been observed following treatment with paraquat, hyperoxia, tumor necrosis factor, interleukin-1 and lipopolysaccharide 6'7.t°'14A6,19. Axonal injury causes various changes in motoneurons: R N A content, uptake of amino acids and protein synthesis increase in motoneurons following axotomy 17, while levels of the molecules related to neural transmission, including choline acetyltransferase and acetylcholinesterase, decrease after this procedure 9. Axotomized motoneurons appear to stop synthesizing their neurotransmitter, acetylcholine, and then appear to increase synthesis of several proteins which are needed during regeneration and axonal sprouting 9. The present study was undertaken to determine whether the synthesis of Mn- and Cu/Zn-SOD

are involved in these motoneuron changes. Thirty-one adult male Wistar rats weighing 200-300 g were used. The animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and were subjected to unilateral transection of the left facial nerve (22 animals) or to unilateral nerve crush (6 animals) 18. Following the operaUons, the animals were killed by decapitation under anesthesia with pentobarbital (50 mg/kg), at 2, 4 and 12 h and i, 4, 7, 28 and 56 days (nerve transected animals) or 4, 7 and 28 days (nerve crushed animals) post-operatively. The brains were immediately excised and frozen on crushed dry ice. Fifteen-~m-thick sections were cut on a cryostat, mounted on silan-coated slides, stored at -70°C, and subjected to in situ hybridization histochemistry. Hybridization procedures with alkaline phosphatase (ALP)-labeled probes were carried out as described previously ~5. The sections were fixed with 4% paraformaldehyde in 0.i M phosphate buffer (PB), p H 7.4, for 20 min, rinsed in the same buffer, and dehydrated in a series of graded ethanols. Hybridization was performed at 37°C with ALP-labeled oligodeoxynucleotide probes for Mn- and Cu/Zn-SOD, containing 50% formamide, 4× SSC, l × Denhardt's solution, 10% dextran sulfate and 15 #g/ml of yeast t R N A . Sections were then washed 4 times at 50°C in l x SSC for 30 min each time and incubated for 48 h in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate kit in 0.1 M Tris buffer, p H 9.5 (Kirkegaard & Perry Lab.). The reaction was stopped by incubation in 0.1 M Tris-HCl

Correspondence H Takagl, First Department of Anatomy, Osaka City Umversaty Me&cal School, Asahlmachl 1-4-54, Abeno-ku, Osaka, 545 Japan.

343 buffer (pH 7.5) containing 10 mM EDTA. The oligodeoxynucleotide probes were complementary to bases 178224 of rat Mn-SOD c D N A 8 and bases 423-471 of rat Cu/Zn-SOD e D N A 5, respectively. ALP was cross-linked to each probe at the thymidine base complementary to the adenosine base, the 193th base of Mn-SOD eDNA or the 445th base of Cu/Zn-SOD. In the competition experiments in which sections were prehybridized with an

excess of non-labeled probes, specific A L P staining following hybridization was virtually absent. For immunohistochemistry, three animals anesthetized with pentobarbital (50 mg/kg) were p erfused transcardially with aldehyde fixative containing 2% paraformaldehyde, 0.2% picric acid and 0.1 M phosphate buffer (pH 7.4). The brains were immersed in 30% sucrose, cut into 30-/~m-thick sections on a cryostat, and subjected to


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Fig. 1. Photormcrographs showing facial motoneurons expressing Mn-SOD mRNA stained by ALP on the operated (B,D,F) and contralateral side (A,C,E) at 1, 7 and 56 days after nerve transection. Bar = 100 ~m.







Fig. 2. Photomicrographs showing facial motoneurons expressing Cu/Zn-SOD mRNA stained by ALP on the operated (B) and unoperated side (A) at 7 days after nerve crush. Bar = 100/~m.

immunohistochemistry as described previously 12A3. The sections were incubated first with rabbit antiserum to purified rat Mn-SOD 13, then with biotinylated anti-rabbit IgG, and finally with ABC complex (Vectastain) 11. Immunoprecipitation was produced by incubation with 0.03% diaminobenzidine tetra-HC1 and 0.005% H 2 0 2 . In the facial nucleus of untreated animals, most motoneurons expressed both Mn-SOD and Cu/Zn-SOD mRNAs. No significant change of either Mn-SOD or Cu/Zn-SOD m R N A was found in motoneurons 2-4 h after axotomy. A slight increase of Mn-SOD m R N A was detected 12 h after transection, and a significant increase was seen in the ipsilateral facial nucleus 1 day after this operation (Fig. 1A,B). This increase in the ipsilateral neurons was most pronounced 4-7 days after transection (Fig. 1C,D), and disappeared 56 days after axotomy (Fig. 1E,F). In contrast, no remarkable change of Cu/ Zn-SOD m R N A was detected in the neurons during any


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period after axotomy or nerve crush (Fig. 2). Crushing the facial nerve resulted in changes in the Mn-SOD m R N A levels in motoneurons that were similar to those observed after nerve transection. The increase of MnSOD m R N A was pronounced 4-7 days after nerve crush, and by 28 days this change disappeared. We examined the changes in Mn-SOD protein in axotomized neurons by immunohistochemistry. Immunoreactivity was significantly increased in the axotomized neurons, and immunostaining was widespread in the dendrites as well as the perikarya (Fig. 3). In the present study an increase of Mn-SOD m R N A levels was seen in the ipsilateral facial motoneurons 12 h-28 days after axotomy, while there was no effect on Cu/Zn-SOD m R N A levels. The peak of this increase was found 4-7 days after axotomy, and by 56 days there was no significant difference between the operated and unoperated sides. Nerve crush resulted in a shorter-term

345 increase of M n - S O D m R N A levels in the facial nucleus,

superoxide radicals 3, which are most probably produced

and by 28 days no significant difference was seen be-

by activation of the respiratory chain following such en-

tween the operated and u n o p e r a t e d sides. This result appears to correspond to the rapid recovery of facial nerve functions after nerve crush, as evidenced by the

hancements of metabolism as glucose uptake, metabolic turnover, and protein synthesis 17.

recovery of whisker m o v e m e n t within 2 weeks. O n the other hand, no functional recovery was observed following nerve transection. O u r results suggest that the induction of M n - S O D , but not C u / Z n - S O D , may be involved in the regeneration and recovery of axotomized motoneurons 17, and in cell protection against the toxicity of

We thank Drs N Tamguchl and K Suzuki (Osaka University) for their generous supply of Mn-SOD antibody This study was supported by Grant-in-Aid for Scientific Research on Pnotity Areas No. 03224-105 from the Ministry of Education, Science and Culture, Japan, and a grant provided by the Senti Life Soence Foundation.

1 Asada, K , Kanematsu, S., Okada, S. and Hayakawa, T., Phylogenlc d~stnbut~on of three types of superox~de &smutase m organisms and in cell organelles In J.V Banmster and H.A.O. Hill (Eds.), Chemtcal and Btochemtcal Aspects of Superoxide and Superoxlde Dtsmutase, Elsewer, Amsterdam, 1980, pp. 136-153. 2 Bannister, J V, Banmster, W.H. and Rotlho, G., Aspects of the structure, function, and apphcations of superox~de &smutase, CRC Crtt Rev. Btochem, 22 (1987) 111-180 3 Fndowch, I , Superoxlde &smutase, Annu Rev. Btochem., 44 (1975) 147-159 4 Gregory, E M. and Fndovich, I., Induction of superoxlde dismutase by molecular oxygen, J Bactenol, 114 (1973) 543-548. 5 Hass, M.A., Igbal, J., Clerch, L B , Frank, L. and Massaro, D., Rat lung Cu,Zn superoxide &smutase' isolation and sequence of full-length cDNA and studies of enzyme induction, J Chn. Invest., 83 (1989) 1241-1246 6 Hassan, H.M., Biosynthesis and regulation of superoxlde chsmutases, Free Radw. Biol Med, 5 (1988) 377-385. 7 Hassan, H.M. and Fridowch, I , Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds, Arch Btochem. Btophys, 196 (1979) 385-395. 8 Ho, Y.-S. and Crapo, J.D., Nucleotlde sequence of cDNAs coding for rat manganese-containing superoxlde d~smutase, Nuclew Acids Res, 15 (1987) 10070. 9 Hoover, D B and Hancock, J.C., Effects of facml nerve transection on acetylcholinesterase, chohne acetyltransferase and [3H]qulnuchdmyl benzllate binding m rat facial nuclei, Neuroscwnce, 15 (1985) 481-487 10 Houssett, B and Junod, A.P., Enzyme response of cultured endothehal cells to hyperoxm, Bull Eur Phystopathol. Respir , 17 (1981) 107-110. 11 Hsu, S, Rame, L. and Franger, H., Use of avidin-b~otln-peroxldase complex (ABC) m lmmunoperoxidase techmque: a comparison between ABC and unlabeled antibody (PAP) pro-

cedures, J. Histochern Cytochem., 29 (1981) 577-580. 12 Inagakl, S, Suzuki, K., Taniguchl, N. and Takagi, H , Locallzation of Mn-superoxlde dlsmutase (Mn-SOD) in chohnergic and somatostatinerg~c neurons in the rat neostnatum, Brain Res, 549 (1991) 174-177. 13 Inagakl, S., Takagi, H., Suzuki, K , Akai, E and Tamguchi, N., Intense ~mmunoreactivity for Mn-superoxide dlsmutase (MnSOD) m cholmerglc and non-chohnerglc neurons m the rat basal forebram, Brain Res., 541 (1991) 354-357 14 Kawaguchl, T., Takeyasu, A , Matsunobu, K , Uda, T., Ishlzawa, M., Suzuki, K., Nishmra, T., Ishikawa, M. and Taniguch~, N , Stimulation of Mn-superoxide dismutase expression by tumor necrosis factor-a' quantitative demonstration of Mn-SOD protein levels m TNF-resistant and sensitive cells by ELISA, Btochem. Biophy. Res Commun., 171 (1990) 1378-1386. 15 K~yama,H., Emson, P C. and Ruth, J., D~stnbution of tyrosme hydroxylase mRNA in the rat central nervous system v~sualized by alkaline phosphatase in sltu hybridization hlstochemistry, Eur J Neurosct., 2 (1990) 512-524. 16 Krall, J Bagley, A.C., Mullenbach, G.T., Hallewell, R.A. and Lynch, R.E., Superoxlde me&ates the toxicity of paraquat for cultured mammahan cells, J Btol. Chem., 263 (1988) 19101914. 17 Lleberman, A.R, The axon reacuon: a review of the principal features of penkaryal responses to axon injury, Int Rev. Neurobtol., 14 (1971) 49-124. 18 Saika, T., Senba, E., Noguchl, K., Sato, M., Yoshida, S., Kubo, T, Matsunaga, T and Tohyama, M., Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor m the rat facml motoneurons, Mol. Brain Res, 9 (1991) 157-160. 19 Visner, G.A., Dougall, W.C., Wilson, J.M., Burr, I.A. and Nick, H.S., Regulation of manganese superoxide dlsmutase by hpopolysaccharide, mterleukin-1, and tumor necrosis factor, J. Btol. Chem., 265 (1990) 2856-2864 ,

zinc superoxide dismutases by the facial nerve transection.

The effects of unilateral nerve transection on manganese and copper/zinc superoxide dismutase (Mn-SOD and Cu/Zn-SOD) mRNA levels in the facial nucleus...
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