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Increased tubulin messenger RNA in the frog retina after optic nerve transection
144
Neuroscience Letters, 86 (1988) 144 146 Elsevier Scientific Publishers Ireland Ltd.
NSL 05202
Increased tubulin messenger R N A in the frog retina after optic nerve transection T o k i h i d e M i z o b u c h i l, 2, Y a s u y u k i Yagi 2, A r i t a k e M i z u n o 1,2, Hiroshi Matsuzaki I and Makoto Matsuda 2 1Department q/'Ophthalmology and :Department of Biochemistry, The Jikei University School ~['Medicine, Tokyo (Japan) (Received 14 October 1987; Revised version received 8 December 1987; Accepted 9 December 1987)
Key word~. Optic nerve; Nerve regeneration; Bullfrog; Axonal transport; Northern blotting: Tubulin messenger R N A The optic nerve of the bullfrog (Rana catesbeiana) was transected and the regeneration process was investigated. The nerve regeneration was confirmed by observing the extension of the axon and by examining the recovery of axonal transport. Following optic nerve transection, the rapid and transient increase of messenger R N A of 7-tubulin gene was observed in the retina, but no specific change of actin messenger R N A was observed.
The neurons of the mammalian central nervous system (CNS) has little ability to regenerate. On the contrary, the CNS neurons of lower vertebrates, such as .frogs and fishes, are able to reinitiate axon extension in response to axonal injury and axon regeneration in these neurons can lead to functional recovery [1]. Following optic nerve crush in goldfish, biochemical changes were observed in the retina, including increased nucleotide uptake and phosphorylation [2]. We confirmed the regeneration of retinotectal fibers following optic nerve transection in the bullfrog and investigated the specific gene expression in the retina. Bullfrogs (250-300 g) were anesthetized with tricaine methanesulfonate (MS 222). The optic nerve was gently separated from other tissues to minimize vascular damage and transected totally between the eye ball and optic chiasma. The frogs were maintained at room temperature for 3 months after the operation. To label axonally transported proteins, we injected 10/~Ci of L-[4,5-3H]leucine (neutralized with trizma base) into the vitreous cavity. One week after the injection, the optic nerve, optic tract and optic tectum were excised under analgesia. Each tissue was solubilized in 1 ml of Protosol (NEN) and the radioactivity of transported proteins was determined by liquid scintillation counting. Correspondence: T. Mizobuchi, Department of Biochemistry, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan. 0304-3940/88/$ 03.50 ~) 1988 Elsevier Scientific Publishers I reland Ltd.
145
A
B
TUBULIN 1
2
3
4
5
6
ACTIN 1
2
3
4
5
6
Ori - -
28 18
Fig. 1. Northern blot analysis of frog retina and rat brain RNA. The optic nerves of bullfrogs were not transected (lane 3) and transected (lanes 445). The retinas of operated frogs were excised at 1 h (lane 4), 2 h (lane 5), and 4 h (lane 6) after transection. The total cellular RNA of retinas was prepared as described above. Rat brain RNA (8/~g, lane 1 and 16/tg, lane 2) and frog retina RNA (8/~g, lanes 3~5) were electrophoresed on a 1% formaldehyde agarose gel, transferred to nitrocellulose membrane, and hybridized to the 32p-labeled ~t-tubulin DNA (A) or actin DNA (B) (specific radioactivity of each probe was about 3 × 109 dprn//~g and about 2 x 107 dpm was used for each hybridization). DNA-RNA hybrids were visualized by autoradiography for 3 days at -70°C.
Three m o n t h s after the operation, the transected region of the optic nerve was observed to heal up to its original outward. W h e n axonal t r a n s p o r t was allowed to proceed for one week after the injection, the labeled proteins were t r a n s p o r t e d to the tectum of a n operated frog a n d the a m o u n t o f their radioactivity was almost the same as that of an u n o p e r a t e d c o n t r o l frog (data n o t shown). The increased labeling o f the t r a n s p o r t e d proteins in the tectum indicates reinitiation o f a x o n extension a n d recovery of axonal t r a n s p o r t from the retinal g a n g l i o n cells [5]. T o assay for specific gene expression in the retina o f the bullfrog, the optic nerve was transected as described above; we excised the retinas at 1, 2, a n d 4 h after optic nerve transection. The total cellular R N A in the retina was isolated by the guanidin i u m / c e s i u m chloride m e t h o d [6]. The R N A in rat b r a i n was also isolated by the same method. These R N A samples were assayed for dot blot or N o r t h e r n blot hybridization to a 32p-labeled ct-tubulin gene a n d actin gene probe*. They were purified by agarose gel electrophoresis a n d digested with EcoRI, restriction enzyme. The *Cloned cDNA of chick brain ~t-tubulinhas lengths of 1.4kb and is inserted into the PstI site of pBR322, while cloned cDNA of Drosophila actin has lengths of 3.6kb and is inserted into the BamHI site of pBR322.
146 D N A fragments were labeled with [~-32p]dCTP by Multiprime D N A labelling system (Amersham). The R N A analysis showed that in frog retina the basal expression o f tubulin m R N A was detected at a lower level than that in rat brain (Fig. 1A, lanes I and 3), tubulin m R N A in the retina reached a m a x i m u m 1-2 h after optic nerve transection and declined to the basal level 4 h after transection (Fig. 1A, lanes 4~6). U n d e r identical conditions, however, no significant changes were detected on the level o f actin m R N A (Fig. IB). Burrell et al. reported that tubulin in translation products o f goldfish retinal poly(A)-containing R N A had continuously increased from 4 days after optic nerve crush [3]. This finding, however, is considered not to be associated with the dramatic expression o f tubulin gene described above because there is a clear discrepancy in the time courses o f these phenomena. Their result is probably interpreted as providing the structural materials necessary for the elaboration o f the new axon. On the contrary, the ral~id and transient expression o f the tubulin gene observed in our study is p r o b a b l y one o f the initial responses of retinal ganglion cells to the axotomy, though its biochemical meaning is not clear yet. Further studies are necessary to elucidate biochemically the discrepancy between the rapid transient expression o f the tubulin gene and the delayed increased production o f tubulin, presumably after tubulingene expression reported by Burrell et al. Such studies are now in progress in our laboratory; they will provide further information regarding the expression o f the tubulin gene in the optic nerve regeneration. The specific expression o f cytoskeleton genes, such as tubulin and actin, is very interesting because it is closely related to nerve proliferation and regeneration [4]. We thank Dr. Ryuhei K a n a m o t o for his generous advice for N o r t h e r n hybridization and Dr. T a k a o Arai for providing the clones o f tubulin (cloned by Dr. M.W. Kirschner) and actin (cloned by Dr. B.J. Bond). 1 Attardi, D.G. and Sperry, R.W., Preferential selection of central pathways by regeneration optic fibers, Exp. Neurol., 7 (1963) 46~34. 2 Burrell, H., Dokas, L. and Agranoff, B.W., RNA metabolism in the goldfish retina during optic nerve regeneration, J. Neurochem., 31 (1978) 289-298. 3 Burrell, H.R., Heacock, A.M., Water, R.D. and Agranoff, B.W., Increased tubulin messenger RNA in the goldfish retina during optic nerve regeneration, Brain Res., 168 (1979) 628 632. 4 Fine, R.E. and Bray, D., Actin in growing nerve cells, Nature New Biol., 234 ( 1971) 115--118. 5 Skene, J.H.P. and Willard, M., Changes in a×onally transported proteins during axon regeneration in toad retinal ganglion cells, J. Cell Biol., 89 (1981) 86-95. 6 Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J. and Goodman, H.M., Rat insulin genes: construction of plasmids containing the coding sequences, Science, 196 (1977) 1313 1319.
Neuroscience Letters, 86 (1988) 144 146 Elsevier Scientific Publishers Ireland Ltd.
NSL 05202
Increased tubulin messenger R N A in the frog retina after optic nerve transection T o k i h i d e M i z o b u c h i l, 2, Y a s u y u k i Yagi 2, A r i t a k e M i z u n o 1,2, Hiroshi Matsuzaki I and Makoto Matsuda 2 1Department q/'Ophthalmology and :Department of Biochemistry, The Jikei University School ~['Medicine, Tokyo (Japan) (Received 14 October 1987; Revised version received 8 December 1987; Accepted 9 December 1987)
Key word~. Optic nerve; Nerve regeneration; Bullfrog; Axonal transport; Northern blotting: Tubulin messenger R N A The optic nerve of the bullfrog (Rana catesbeiana) was transected and the regeneration process was investigated. The nerve regeneration was confirmed by observing the extension of the axon and by examining the recovery of axonal transport. Following optic nerve transection, the rapid and transient increase of messenger R N A of 7-tubulin gene was observed in the retina, but no specific change of actin messenger R N A was observed.
The neurons of the mammalian central nervous system (CNS) has little ability to regenerate. On the contrary, the CNS neurons of lower vertebrates, such as .frogs and fishes, are able to reinitiate axon extension in response to axonal injury and axon regeneration in these neurons can lead to functional recovery [1]. Following optic nerve crush in goldfish, biochemical changes were observed in the retina, including increased nucleotide uptake and phosphorylation [2]. We confirmed the regeneration of retinotectal fibers following optic nerve transection in the bullfrog and investigated the specific gene expression in the retina. Bullfrogs (250-300 g) were anesthetized with tricaine methanesulfonate (MS 222). The optic nerve was gently separated from other tissues to minimize vascular damage and transected totally between the eye ball and optic chiasma. The frogs were maintained at room temperature for 3 months after the operation. To label axonally transported proteins, we injected 10/~Ci of L-[4,5-3H]leucine (neutralized with trizma base) into the vitreous cavity. One week after the injection, the optic nerve, optic tract and optic tectum were excised under analgesia. Each tissue was solubilized in 1 ml of Protosol (NEN) and the radioactivity of transported proteins was determined by liquid scintillation counting. Correspondence: T. Mizobuchi, Department of Biochemistry, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan. 0304-3940/88/$ 03.50 ~) 1988 Elsevier Scientific Publishers I reland Ltd.
145
A
B
TUBULIN 1
2
3
4
5
6
ACTIN 1
2
3
4
5
6
Ori - -
28 18
Fig. 1. Northern blot analysis of frog retina and rat brain RNA. The optic nerves of bullfrogs were not transected (lane 3) and transected (lanes 445). The retinas of operated frogs were excised at 1 h (lane 4), 2 h (lane 5), and 4 h (lane 6) after transection. The total cellular RNA of retinas was prepared as described above. Rat brain RNA (8/~g, lane 1 and 16/tg, lane 2) and frog retina RNA (8/~g, lanes 3~5) were electrophoresed on a 1% formaldehyde agarose gel, transferred to nitrocellulose membrane, and hybridized to the 32p-labeled ~t-tubulin DNA (A) or actin DNA (B) (specific radioactivity of each probe was about 3 × 109 dprn//~g and about 2 x 107 dpm was used for each hybridization). DNA-RNA hybrids were visualized by autoradiography for 3 days at -70°C.
Three m o n t h s after the operation, the transected region of the optic nerve was observed to heal up to its original outward. W h e n axonal t r a n s p o r t was allowed to proceed for one week after the injection, the labeled proteins were t r a n s p o r t e d to the tectum of a n operated frog a n d the a m o u n t o f their radioactivity was almost the same as that of an u n o p e r a t e d c o n t r o l frog (data n o t shown). The increased labeling o f the t r a n s p o r t e d proteins in the tectum indicates reinitiation o f a x o n extension a n d recovery of axonal t r a n s p o r t from the retinal g a n g l i o n cells [5]. T o assay for specific gene expression in the retina o f the bullfrog, the optic nerve was transected as described above; we excised the retinas at 1, 2, a n d 4 h after optic nerve transection. The total cellular R N A in the retina was isolated by the guanidin i u m / c e s i u m chloride m e t h o d [6]. The R N A in rat b r a i n was also isolated by the same method. These R N A samples were assayed for dot blot or N o r t h e r n blot hybridization to a 32p-labeled ct-tubulin gene a n d actin gene probe*. They were purified by agarose gel electrophoresis a n d digested with EcoRI, restriction enzyme. The *Cloned cDNA of chick brain ~t-tubulinhas lengths of 1.4kb and is inserted into the PstI site of pBR322, while cloned cDNA of Drosophila actin has lengths of 3.6kb and is inserted into the BamHI site of pBR322.
146 D N A fragments were labeled with [~-32p]dCTP by Multiprime D N A labelling system (Amersham). The R N A analysis showed that in frog retina the basal expression o f tubulin m R N A was detected at a lower level than that in rat brain (Fig. 1A, lanes I and 3), tubulin m R N A in the retina reached a m a x i m u m 1-2 h after optic nerve transection and declined to the basal level 4 h after transection (Fig. 1A, lanes 4~6). U n d e r identical conditions, however, no significant changes were detected on the level o f actin m R N A (Fig. IB). Burrell et al. reported that tubulin in translation products o f goldfish retinal poly(A)-containing R N A had continuously increased from 4 days after optic nerve crush [3]. This finding, however, is considered not to be associated with the dramatic expression o f tubulin gene described above because there is a clear discrepancy in the time courses o f these phenomena. Their result is probably interpreted as providing the structural materials necessary for the elaboration o f the new axon. On the contrary, the ral~id and transient expression o f the tubulin gene observed in our study is p r o b a b l y one o f the initial responses of retinal ganglion cells to the axotomy, though its biochemical meaning is not clear yet. Further studies are necessary to elucidate biochemically the discrepancy between the rapid transient expression o f the tubulin gene and the delayed increased production o f tubulin, presumably after tubulingene expression reported by Burrell et al. Such studies are now in progress in our laboratory; they will provide further information regarding the expression o f the tubulin gene in the optic nerve regeneration. The specific expression o f cytoskeleton genes, such as tubulin and actin, is very interesting because it is closely related to nerve proliferation and regeneration [4]. We thank Dr. Ryuhei K a n a m o t o for his generous advice for N o r t h e r n hybridization and Dr. T a k a o Arai for providing the clones o f tubulin (cloned by Dr. M.W. Kirschner) and actin (cloned by Dr. B.J. Bond). 1 Attardi, D.G. and Sperry, R.W., Preferential selection of central pathways by regeneration optic fibers, Exp. Neurol., 7 (1963) 46~34. 2 Burrell, H., Dokas, L. and Agranoff, B.W., RNA metabolism in the goldfish retina during optic nerve regeneration, J. Neurochem., 31 (1978) 289-298. 3 Burrell, H.R., Heacock, A.M., Water, R.D. and Agranoff, B.W., Increased tubulin messenger RNA in the goldfish retina during optic nerve regeneration, Brain Res., 168 (1979) 628 632. 4 Fine, R.E. and Bray, D., Actin in growing nerve cells, Nature New Biol., 234 ( 1971) 115--118. 5 Skene, J.H.P. and Willard, M., Changes in a×onally transported proteins during axon regeneration in toad retinal ganglion cells, J. Cell Biol., 89 (1981) 86-95. 6 Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J. and Goodman, H.M., Rat insulin genes: construction of plasmids containing the coding sequences, Science, 196 (1977) 1313 1319.