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Polyamines promote regeneration of injured axons of cultured rat hippocampal neurons
BRAIN RESEARCH ELSEVIER
Brain Research 673 (1995) 233-241
Research report
Polyamines promote regeneration of injured axons of cultured rat hippocampal neurons Peng-jiang Chu, Hiroshi Saito, Kazuho Abe * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan
Accepted 22 November 1994
Abstract
Axons of cultured rat hippocampal neurons were injured by local irradiation of laser beam, and the effects of spermine, spermidine and putrescine on neurite regeneration following axonal injury were investigated. The axonal growth was stopped by laser irradiation, but addition of spermine remarkably promoted the axonal re-elongation from the injured site. Spermine affected neither the neurite branching at proximal part of injured axons nor the growth of uninjured dendrites. The effect of spermine was concentration dependent and seen maximally at a concentration of 10 8 M. Spermidine and putrescine also promoted the axonal re-elongation in a concentration-dependent manner. The effects of three polyamines were very similar, and no additivity was observed when maximally effective concentrations of polyamines were added together, suggesting that they act through a common mechanism. Unlike polyamines, basic fibroblast growth factor (bFGF) did not promote the axonal re-elongation from the injured site, but rather stimulated the formation of axonal branches at proximal part of injured axons, supporting that the promotion of axonal re-elongation is a specifc action of polyamines. Concomitant addition of spermine and bFGF additively or synergistically promoted both the axonal re-elongation from the injured site and the branch formation at proximal part of injured axons. These data suggest that polyamines have a capability of promoting axonal regeneration of brain neurons after lesioning. Keywords: Spermine; Spermidine; Putrescine; Neurite regeneration; Basic fibroblast growth factor; Cultured hippocampal neuron
1. Introduction
The endogenous polyamines, spermine, spermidine and putrescine, are known to play important roles in proliferation and differentiation of many types of cells [11,20,27]. However, the functions of the endogenous polyamines on brain neurons have not been well understood. We have recently reported that spermine, but not spermidine and putrescine, supported the survival of primary cultured hippocampal neurons and that spermine as well as spermidine and putrescine promoted the neurite outgrowth of cultured rat hippocampal neurons [1,6], suggesting that polyamines function as neurotrophic factors for brain neurons. Many proteinous neurotrophic factors have been reported, but they have several problems in clinical use,
* Corresponding author. Fax: (81) (3) 3815-4603. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01419-1
e.g., very low bioavailability, difficult developement of more active analogues, possible side effects, etc. The neurotrophic action of polyamines with simple chemical structures is of great interest in relation to the developement of therapeutic drugs for neurodegenerative disorders such as Alzheimer's disease. Furthermore, it has been reported that the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, and the levels of endogenous polyamines in the brain are increased following transient cerebral ischemia [8,19], implying that polyamines may play some role in the brain under pathological conditions. Whether or not polyamines exert effects on damaged neurons is an important subject to clarify a role for polyamines in neural pathologies. We have recently developed a simple assay system in which the growth cone of the axon of cultured neurons was injured by local irradiation of laser beam and the effects of drugs were evaluated for axonal re-elonga-
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
234
(1) Axon length from soma to injured site
(2) Regenerated axon length
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2. Materials and methods
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We used the ACAS (adherent cell analysis system) 470 Work Station (Meridian) that consists of a computer, an inverted microscope and a laser system. The
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Fig. 1. Morphological parameters measured to quantify possible responses of neurons following axonal lesioning. First (1) the axon length from soma to injured site was measured from the photographs taken immediately after laser irradiation, and it was confimed that this value was not different among the tested groups, since the distance of the lesioned site from the soma might influence the regenerative response of neurons. (2) Regenerated axon length from injured site, (3) the number of branch points in regenerated axon and (4) total length of regenerated axon including branches were measured as parameters of axonal regeneration from lesioned site. Changes in branches at proximal part of injured axons were quantified by measuring (5) the number of branch points and (6) the total length of axonal branches. Changes in uninjured neurites (dendrites) were quantified by measuring (7) the number of primary neurites directly emanating from the soma and (8) the total length of dendrites. White arrowheads indicate the site injured by laser irradiation. The number of neurites or branch points was counted as illustrated. Neurite length corresponded to the length of lines drawn in bold strokes.
tion or other morphological changes in the partially damaged neurons [16]. By using this assay system, in the present study, we investigated the effects of spermine, spermidine and putrescine on neurite regeneration of injured brain neurons in vitro, and found that polyamines strongly promoted the axonal regeneration. Moreover, the effects of polyamines were compared with that of basic fibroblast growth factor (bFGF), a neurotrophic factor for brain neurons [2,17,28], since we have previously found that b F G F promoted the formation of axonal branches in cultured hippocampal neurons [3,16]. The role of polyamines in neuronal regeneration will be discussed in comparison with bFGF.
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Fig. 2. The influence of laser irradiation to a site proximal to axonal growth cone on morphology of cultured rat hippocampal neurons. The axon length (A), the number of branch points in proximal part of axon (B), the total length of branches in proximal part of axon (C), the number of primary neurites per soma (D) and the total length of dendrites (E) were compared between intact neurons (Intact, n = 23) and lesioned neurons (Laser, n = 21) 0 (white columns), 24 (hatched columns) and 48 h (solid black columns) after laser irradiation. The data are represented as the mean _+S.E.M. Asterisks indicate significant differences from the values in intact group: * *P < 0.01; Dunnett's test.
P.-j. Chu et aL / Brain Research 673 (1995) 233-241
computer served to memorize the cell position on the microscope stage and control the intensity and duration of Argon laser (488 nm) irradiation, b F G F used in the present study is an acid-resistant mutein of human bFGF, CS23 (a generous gift from Takada Chemical Industries, Ltd., Osaka, Japan). The activity of CS23 on brain neurons is virtually the same as that of wild type of human b F G F [2]. Dissociated hippocampal neurons were prepared from 18-day-old embryos of Wistar rats as described in our previous paper [2] and plated on polylysine-coated culture dish at a density of 2500 cells/cm 2. The culture dish was prepared from a 35-mm Petri dish by attaching a special glass coverslip to the inner surface with silicone greace [16]. The special glass coverslip was sealed with a film that absorbs laser beam and converts laser beam to heat. When laser beam was irradiated, the neurites were virtually damaged by heat. After
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incubation in serum-containing medium for 24 h, the medium was changed into serum-free modified Eagle's medium supplemented with transferrin, insulin and progesterone [6]. While cultured in the serum-free medium for a further 24 h, hippocampal pyramidal neurons acquired their stereotypical morphology with one long process and several short processes. By morphological and immunocytological criteria, the long process and several short processes have been identified as axon and dendrites, respectively [4,5,14]. The term 'neurites' is used to include both axons and dendrites. The pyramidal-like neurons that had established axons and dendrites and were free from contact with other cells were selected in each group, and the laser beam (output 40 mW; acoust-optic-modulator, 35%) was delivered to a site proximal to the growth cone of the axon for 1 s. This moderate condition of laser irradiation did not significantly affect the survival
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Fig. 3. Representative photomicrographs showing cultured rat hippocampal neurons immediately following axotomy (0 h) and 24 or 48 h after axotomy in the control medium (A) or in the medium with added 10 8 M spermine (B). Arrows indicate the sites injured by laser irradiation. Bars, 100/zm.
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
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Fig. 4. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on regenerated axon length from injured site ( A - C ) and the number of branch points in regenerated axons ( D - F ) following laser irradiation. Hatched and solid black columns show the data at times 24 and 48 h after lesioning, respectively. The data are represented as the mean + S.E.M. (n = 21-25). Asterisks indicate significant differences from the values in group which received axonal lesions but was not treated with polyamines (Cont): * *P < 0.01; Dunnett's test.
of neurons. The cells were photographed immediately after laser irradiation and then drugs were added to the culture medium. The same cells were photographed 24 and 48 h after addition of drugs. If the selected neurons had died within 48 h, the data of the cells were omitted. Measurement of morphological parameters was made by tracing the photographs on a digitizing tablet. Possible responses of neurons following axonal injury are (1) neurite regeneration from injured site, (2) changes in axonal branches at proximal part of injured
axon, and (3) changes in uninjured dendrites. Furthermore, neuritic shape is formed by two different processes, 'elongation' and 'branching'. Therefore, we measured the following morphological parameters: (1) axon length from soma to injured site, (2) regenerated axon length from injured site, (3) the number of branch points in regenerated axon, (4) total length of regenerated axon including branches, (5) the number of branch points in proximal part of injured axon, (6) total length of branches in proximal part of injured axon, (7) the number of primary neurites per soma, and 8) total
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Fig. 5. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on the number of branch points ( A - C ) and total length of branches ( D - F ) in proximal part of injured axons. White, hatched and solid black columns show the data at times 0, 24 and 48 h after lesioning, respectively. The data are represented as the mean + S.E.M. (n = 21-25).
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
from soma (Fig. 6A) and total length of uninjured dendrites (Fig. 6D) were not affected by the addition of spermine. The axonal regeneration-promoting effect of spermine was concentration dependent and seen maximally at 10 -8 M. Spermidine and putrescine also promoted the re-elongation of injured axons with the maximum effect at a concentration of 10 -8 M (Fig. 4B and C). The growth of branches in proximal part of injured axons and uninjured dendrites were not affected by spermidine or putrescine (Fig. 5B,C,E,F and Fig. 6B,C,E,F). The effects of three polyamines were very similar in terms of both the effective concentrations and the patterns of morphological changes induced by them, implying that all three polyamines may act through a common mechanism. To examine this possibility, we investigated whether or not the additivity was seen when polyamines were added together. As shown in Fig. 7A, axonal re-elongation induced by spermine, spermidine and putrescine at a maximally effective concentration (10 -s M) were virtually the same, and no additivity was seen when spermine was added together with spermidine or putrescine. Concomitant addition of spermine and spermidine or putrescine affected neither the branches in proximal part of injured axons (Fig. 7B) nor the uninjured dendrites (Fig. 7C and D). Furthermore, the effect of spermine was compared with that of bFGF. Addition of b F G F (1 ng/ml), unlike spermine, did not promote the re-elongation of injured axons (Fig. 8B and C), but the number of branch points and total length of branches in proximal part of injured axons were dramatically increased by b F G F (Fig. 8E and F). The numbers of primary neu-
length of uninjured dendrites. Definition of each morphological parameter is illustrated in detail in Fig. 1.
3. Results
First we checked the influence of laser irradiation on morphology of cultured hippocampal neurons. Axonal elongation ceased following the laser irradiation, but the growth of branches at proximal part of axon, the number of neurites per soma and dendritic growth were not affected by the laser irradiation (Figs. 2 and 3A). Next the effects of polyamines on the morphological changes following axonal lesioning were investigated. Since the distance of the injured site from the soma may influence the regenerative response of neurons [10], we confirmed that the axon length from soma to the injured site was not significantly different among the tested groups (data not shown). Fig. 3B shows micrographs of a representative neuron cultured in the medium with added spermine. The results of quantitative analyses in many neurons are shown in Figs. 4-6. Addition of spermine greatly promoted the re-elongation of axons from the injured site (Fig. 4A). No significant increase in branch points in regenerated axons (Fig. 4D) means that injured axons extended without bifurcating. Spermine did not affect the number of branch points in proximal part of injured axons (Fig. 5A). Total length of branches in proximal part of injured axon tended to be increased by the presence of spermine, but the effect was not statistically significant (Fig. 5D). The number of primary neurites emanating
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Fig. 6. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on the n u m b e r of primary neurites per soma ( A - C ) and the total length of uninjured dendrites ( D - F ) . White, hatched and solid columns show the data at times 0, 24 and 48 h after lesioning, respectively. The data are represented as the m e a n +. S.E.M. (n = 21-25).
238
P.-j. Chu et aL / B r a i n Research 673 (1995) 233-241
rites per soma (Fig. 8G) and total length of uninjured dendrites (Fig. 8H) were not affected by bFGF. When spermine (10 -8 M) and b F G F (1 n g / m l ) were added together, both the axonal re-elongation from injured site and the growth of branches in proximal part of injured axons were promoted (Fig. 8B,C and F). Interestingly, the number of branch points in regenerated axons, which was not significantly affected by spermine or b F G F alone, was significantly increased by concomitant addition of spermine and b F G F (Fig. 8D). The number of primary neurites per soma and the growth of uninjured neurites were not altered by simultaneous addition of spermine and b F G F (Fig. 8G and H). The effects of spermine and b F G F are schematically summarized in Fig. 9.
Therefore, it is probable that three polyamines act through a common mechanism. Polyamines are known to potentiate the N-methyl-D-aspartate (NMDA) receptor-mediated responses [7,18,21,26,29]. However, Mattson et al. [14] have reported that neurite outgrowth of cultured hippocampal neurons was reduced
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4. D i s c u s s i o n
The main finding in the present study is that spermine, spermidine and putrescine were very effective in promoting the re-elongation of injured axons of cultured rat hippocampal neurons. First, the following data support that the effects of polyamines observed in the present study is not due to a secondary effect of enhanced survival of neurons: (1) the moderate condition of laser irradiation employed in the present study stopped the axonal growth, but did not affect the survival and other morphological parameters of neurons; (2) we have previously observed that spermine was effective in supporting the survival of cultured rat hippocampal neurons but spermidine and putrescine had no effect [1,6], whereas, in the present study, three polyamines similarly promoted the axonal re-elongation. Furthermore, the re-elongation of injured axons of neurons which once grew and differentiated in culture seems to be, at least in part, different from the initial phase of growth of dissociated neurons in monolayer culture. For example, interleukin-2 promoted axonal elongation of dissociated hippocampal neurons in normal culture condition [25], but did not promote re-elongation of axons injured by laser irradiation [our unpublished observation]. On the other hand, polyamines more effectively promoted the re-elongation of injured axons than the axonal elongation in normal culture condition [6]. Therefore, the present result suggests that polyamines have a capability of promoting axonal regeneration of brain neurons after lesioning. The mechanism by which polyamines promote the axonal re-elongation is not yet clear. The effects of spermine, spermidine and putrescine were very similar in terms of both the mode of action and effective concentrations, and the concomitant addition of spermine and spermidine or putrescine at a maximally effective concentration showed no additivie effect.
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Fig. 7. No additivity of the effects of spermine (SPN), spermidine (SPD) and putrescine (PUT) on axonal regeneration followinglesioning. Polyamines at a maximally effective concentration (10 s M) were added alone or concomitantly, and the effects on regenerated axon length from injured site (A), the number of branch points in proximal part of injured axons (B), the number of primary neurites per soma (C) and the total length of uninjured dendrites (D) were compared. The date are represented as mean _+S.E.M. (n = 20-24). Asterisks indicate significant differences from the value of control group: * *P < 0.01; Dunnett's test.
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
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Fig. 8. Comparison of the effects of spermine (SPN) and b F G F on neuronal morphology following axonal lesioning. Spermine (10 -8 M) and b F G F (1 n g / m l ) were added alone or concomitantly immediately after laser irradiation, and the morphological parameters were measured at times 0 (white columns), 24 (hatched columns) and 48 h (solid black columns) after addition of drugs. The data are represented as the m e a n + S.E.M. (n = 21-23). Asterisks indicate significant differences from the value of control group: * *P < 0.01; D u n n e t t ' s test.
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Fig. 9. Summary of the effects of spermine and bFGF on neurite regeneratoin following axonal lesioning. Spermine selectively promoted the axonal re-elongation from injured site, while bFGF predominantly stimulated the formation of axonal branches at proximal part of injured axons. Uninjured dendrites were affected by neither spermine nor bFGF. Concomitant addition of spermine and bFGF promoted both the axonal re-elongation from injured site and the axonal branch formation at proximal part of injured axons. Furthermore, branching of regenerated axons occurred by synergistic action of spermine and bFGF.
by kainate and quisqualate but not by N M D A , suggesting that N M D A receptors are not involved in regulating neurite outgrowth. Moreover, putrescine is virtually inactive in potentiating the activation of N M D A receptor channel complex [7,23,24], but promoted the axonal re-elongation as well as spermine and spermidine. Therefore, it is unlikely that polyamines promote the axonal re-elongation by potentiating N M D A response. Alternatively, it is known that polyamines interact with D N A or R N A and stimulate protein biosynthesis in various types of cells [27]. Therefore, it is possible that polyamines regulate the protein synthesis associated with the neuronal regeneration. Polyamines did not affect the growth of branches in proximal part of injured axons and uninjured dendrites. The reason why polyamines promotes predominantly the axonal reelongation of injured axons is not clear, but it is possible that polyamines selectively regulate some cytoskelton component associated with axonal regeneration. In addition, it is also possible that the polyamines affect Ca 2+ homeostasis in hippocampal neurons, for Ca 2+ plays a key role in neurite outgrowth and regeneration
240
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
[12,15]. F u r t h e r investigations a b o u t the m e c h a n i s m of action of the p o l y a m i n e s are b e i n g c o n d u c t e d in o u r laboratory. T h e specificity of the action of p o l y a m i n e s was conspicuous by a c o m p a r i s o n with the effect of b F G F . It has b e e n r e p o r t e d that b F G F p r o m o t e d the survival [2,17,28] a n d n e u r i t e growth of c u l t u r e d b r a i n n e u r o n s [3,15] a n d that b F G F increases in n e u r a l tissues of chemically or m e c h a n i c a l l y lesioned b r a i n [9,13,22], suggesting that b F G F may be also involved in the n e u r o n a l r e g e n e r a t i o n in pathological conditions. U n like polyamines, b F G F had n o capability to p r o m o t e the axonal r e - e l o n g a t i o n from i n j u r e d site, s u p p o r t i n g that the p r o m o t i o n of the axonal r e - e l o n g a t i o n is a specific action of polyamines. Instead, b F G F stimulated the b r a n c h f o r m a t i o n at proximal part of i n j u r e d axons, c o n s i s t e n t with o u r previous observation [16]. S p e r m i n e a n d b F G F seem to rescue the i n j u r e d n e u r o n s t h r o u g h different strategies: s p e r m i n e promotes axonal r e - e l o n g a t i o n from i n j u r e d site, while b F G F stimulates the f o r m a t i o n of axonal b r a n c h e s at proximal part of i n j u r e d axon. W h e n s p e r m i n e a n d b F G F were a d d e d together, both axonal r e - e l o n g a t i o n a n d b r a n c h i n g were p r o m o t e d , suggesting that axonal r e - e l o n g a t i o n and b r a n c h i n g are i n d e p e n d e n t l y regulated by different factors or m e c h a n i s m s . Interestingly, the n u m b e r of b r a n c h points in r e g e n e r a t e d axons was significantly increased by synergistic action of s p e r m i n e a n d b F G F . This effect a p p e a r s to result from that axons r e g e n e r a t e d by the action of s p e r m i n e were f u r t h e r s t i m u l a t e d to bifurcate by b F G F . T h e s e data d e m o n s t r a t e that the c o m b i n e d use of m o r e t h a n o n e drugs that act t h r o u g h different m e c h a n i s m s has the a d v a n t a g e of m o r e effectively p r o m o t i n g the recovery following n e u r o n a l injury. In conclusion, we have shown for the first time that s p e r m i n e , s p e r m i d i n e a n d p u t r e s c i n e strongly a n d selectively p r o m o t e the r e - e l o n g a t i o n of axons of cult u r e d h i p p o c a m p a l n e u r o n s following axonal lesions. It is possible that p o l y a m i n e s play a role in n e u r o n a l r e g e n e r a t i o n following b r a i n injury. I n addition, conc o m i t a n t a d d i t i o n of s p e r m i n e a n d b F G F additively or synergistically p r o m o t e d the axonal re-growth from inj u r e d site a n d the b r a n c h f o r m a t i o n at proximal part of i n j u r e d axons, suggesting the a d v a n t a g e of c o m b i n e d use of m o r e t h a n o n e drugs for t h e r a p y of n e u r o d e g e n erative disorders.
Acknowledgements T h e a u t h o r s are grateful to T a k e d a C h e m i c a l I n d u s tries, Ltd. for the g e n e r o u s gift of b F G F .
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[19]
[20] [21]
[22]
[23]
[24]
potentiate responses of N-methyl-D-aspartate receptors expressed in Xenopus oocytes, Proc. Natl., Acad. Sci. USA., 87 (1990) 9971-9974. Paschen, W., Rohn, G., Meese, C.O., Djuricic, B. and Schumidt-Kastner, R., Polyamine metabolism in reversible cerebral ischemia: effect of a-difluoromethylornithine, Brain Res., 453 (1988) 9-16. Pegg, A.E., Recent advances in the biochemistry of polyamines in eukaryotes, Biochern. J., 234 (1986) 249-262. Ransom, R.W. and Stec, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptor-ion channel complex by L-glutamate, glycine, and polyamine, Z Neurochem., 51 (1988) 830-836. Riva, M.A., Gale, K. and Mochetti, I., Basic fibroblast growth factor mRNA increases in specific brain regions following convulsive seizures, Mol. Brain Res., 15 (1992) 311-318. Romano, C., Williams, K. and Molinoff, P.B., Polyamines modulate the binding of [3H]MK-801 to the solubilized N-methyl-Daspartate receptor, J. Neurochem., 57 (1991) 811-818. Sacaan, A.I. and Johnson, K.M., Characterization of the stimu-
[25]
[26]
[27] [28]
[29]
241
latory and inhibitory effects of polyamines on [3H]N-(1[thienyl]cyclohexyl) piperidine binding to the N-methyl-D-aspartate receptor ionophore complex, Mol. Pharmacol., 37 (1990) 572-577. Sarder, M., Saito, H. and Abe, K., Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain, Brain Res., 625 (1993) 347-350. Sporsen, T.S. and Woodruff, G.N., Polyamines potentiate NMDA induced whole-cell currents in cultured striatal neurons, Eur. J. Pharmacol., 179 (1990) 477-478. Tabor, C.W. and Tabor, H., Polyamines, Annu. Rev. Biochem., 53 (1984) 749-790. Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R., Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension, Proc. Natl. Acad. Sci. USA., 83 (1986) 3012-3016. Williams, K., Dawson, V.L., Romano, C., Dichter, M.A. and Molinoff, P.B., Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of NMDA receptor, Neuron, 5 (1990) 199-208.
Brain Research 673 (1995) 233-241
Research report
Polyamines promote regeneration of injured axons of cultured rat hippocampal neurons Peng-jiang Chu, Hiroshi Saito, Kazuho Abe * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113, Japan
Accepted 22 November 1994
Abstract
Axons of cultured rat hippocampal neurons were injured by local irradiation of laser beam, and the effects of spermine, spermidine and putrescine on neurite regeneration following axonal injury were investigated. The axonal growth was stopped by laser irradiation, but addition of spermine remarkably promoted the axonal re-elongation from the injured site. Spermine affected neither the neurite branching at proximal part of injured axons nor the growth of uninjured dendrites. The effect of spermine was concentration dependent and seen maximally at a concentration of 10 8 M. Spermidine and putrescine also promoted the axonal re-elongation in a concentration-dependent manner. The effects of three polyamines were very similar, and no additivity was observed when maximally effective concentrations of polyamines were added together, suggesting that they act through a common mechanism. Unlike polyamines, basic fibroblast growth factor (bFGF) did not promote the axonal re-elongation from the injured site, but rather stimulated the formation of axonal branches at proximal part of injured axons, supporting that the promotion of axonal re-elongation is a specifc action of polyamines. Concomitant addition of spermine and bFGF additively or synergistically promoted both the axonal re-elongation from the injured site and the branch formation at proximal part of injured axons. These data suggest that polyamines have a capability of promoting axonal regeneration of brain neurons after lesioning. Keywords: Spermine; Spermidine; Putrescine; Neurite regeneration; Basic fibroblast growth factor; Cultured hippocampal neuron
1. Introduction
The endogenous polyamines, spermine, spermidine and putrescine, are known to play important roles in proliferation and differentiation of many types of cells [11,20,27]. However, the functions of the endogenous polyamines on brain neurons have not been well understood. We have recently reported that spermine, but not spermidine and putrescine, supported the survival of primary cultured hippocampal neurons and that spermine as well as spermidine and putrescine promoted the neurite outgrowth of cultured rat hippocampal neurons [1,6], suggesting that polyamines function as neurotrophic factors for brain neurons. Many proteinous neurotrophic factors have been reported, but they have several problems in clinical use,
* Corresponding author. Fax: (81) (3) 3815-4603. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01419-1
e.g., very low bioavailability, difficult developement of more active analogues, possible side effects, etc. The neurotrophic action of polyamines with simple chemical structures is of great interest in relation to the developement of therapeutic drugs for neurodegenerative disorders such as Alzheimer's disease. Furthermore, it has been reported that the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, and the levels of endogenous polyamines in the brain are increased following transient cerebral ischemia [8,19], implying that polyamines may play some role in the brain under pathological conditions. Whether or not polyamines exert effects on damaged neurons is an important subject to clarify a role for polyamines in neural pathologies. We have recently developed a simple assay system in which the growth cone of the axon of cultured neurons was injured by local irradiation of laser beam and the effects of drugs were evaluated for axonal re-elonga-
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
234
(1) Axon length from soma to injured site
(2) Regenerated axon length
v
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2. Materials and methods
...
We used the ACAS (adherent cell analysis system) 470 Work Station (Meridian) that consists of a computer, an inverted microscope and a laser system. The
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(3) No. of branch points in regenerated axon (4) Total length of regenerated axon including branches
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.... ~,
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:,
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(5) No. of branch points in proximal part of axon
0 (6) Total length of axonal branches
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(7) No. of primary neuriteSpersoma
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~
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1
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Fig. 1. Morphological parameters measured to quantify possible responses of neurons following axonal lesioning. First (1) the axon length from soma to injured site was measured from the photographs taken immediately after laser irradiation, and it was confimed that this value was not different among the tested groups, since the distance of the lesioned site from the soma might influence the regenerative response of neurons. (2) Regenerated axon length from injured site, (3) the number of branch points in regenerated axon and (4) total length of regenerated axon including branches were measured as parameters of axonal regeneration from lesioned site. Changes in branches at proximal part of injured axons were quantified by measuring (5) the number of branch points and (6) the total length of axonal branches. Changes in uninjured neurites (dendrites) were quantified by measuring (7) the number of primary neurites directly emanating from the soma and (8) the total length of dendrites. White arrowheads indicate the site injured by laser irradiation. The number of neurites or branch points was counted as illustrated. Neurite length corresponded to the length of lines drawn in bold strokes.
tion or other morphological changes in the partially damaged neurons [16]. By using this assay system, in the present study, we investigated the effects of spermine, spermidine and putrescine on neurite regeneration of injured brain neurons in vitro, and found that polyamines strongly promoted the axonal regeneration. Moreover, the effects of polyamines were compared with that of basic fibroblast growth factor (bFGF), a neurotrophic factor for brain neurons [2,17,28], since we have previously found that b F G F promoted the formation of axonal branches in cultured hippocampal neurons [3,16]. The role of polyamines in neuronal regeneration will be discussed in comparison with bFGF.
_~ " ~ x
25
o o o
-
-
5 E~
o "-I 0
--
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Fig. 2. The influence of laser irradiation to a site proximal to axonal growth cone on morphology of cultured rat hippocampal neurons. The axon length (A), the number of branch points in proximal part of axon (B), the total length of branches in proximal part of axon (C), the number of primary neurites per soma (D) and the total length of dendrites (E) were compared between intact neurons (Intact, n = 23) and lesioned neurons (Laser, n = 21) 0 (white columns), 24 (hatched columns) and 48 h (solid black columns) after laser irradiation. The data are represented as the mean _+S.E.M. Asterisks indicate significant differences from the values in intact group: * *P < 0.01; Dunnett's test.
P.-j. Chu et aL / Brain Research 673 (1995) 233-241
computer served to memorize the cell position on the microscope stage and control the intensity and duration of Argon laser (488 nm) irradiation, b F G F used in the present study is an acid-resistant mutein of human bFGF, CS23 (a generous gift from Takada Chemical Industries, Ltd., Osaka, Japan). The activity of CS23 on brain neurons is virtually the same as that of wild type of human b F G F [2]. Dissociated hippocampal neurons were prepared from 18-day-old embryos of Wistar rats as described in our previous paper [2] and plated on polylysine-coated culture dish at a density of 2500 cells/cm 2. The culture dish was prepared from a 35-mm Petri dish by attaching a special glass coverslip to the inner surface with silicone greace [16]. The special glass coverslip was sealed with a film that absorbs laser beam and converts laser beam to heat. When laser beam was irradiated, the neurites were virtually damaged by heat. After
A
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Control
235
incubation in serum-containing medium for 24 h, the medium was changed into serum-free modified Eagle's medium supplemented with transferrin, insulin and progesterone [6]. While cultured in the serum-free medium for a further 24 h, hippocampal pyramidal neurons acquired their stereotypical morphology with one long process and several short processes. By morphological and immunocytological criteria, the long process and several short processes have been identified as axon and dendrites, respectively [4,5,14]. The term 'neurites' is used to include both axons and dendrites. The pyramidal-like neurons that had established axons and dendrites and were free from contact with other cells were selected in each group, and the laser beam (output 40 mW; acoust-optic-modulator, 35%) was delivered to a site proximal to the growth cone of the axon for 1 s. This moderate condition of laser irradiation did not significantly affect the survival
B
Spermine
nil
24h
48 h
Fig. 3. Representative photomicrographs showing cultured rat hippocampal neurons immediately following axotomy (0 h) and 24 or 48 h after axotomy in the control medium (A) or in the medium with added 10 8 M spermine (B). Arrows indicate the sites injured by laser irradiation. Bars, 100/zm.
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
236
100
100
100
0
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0
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9 8 7 Spermlne (-log.M)
i
0
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9
8
7
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9 8 7 Putresclne (Jog.M)
Fig. 4. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on regenerated axon length from injured site ( A - C ) and the number of branch points in regenerated axons ( D - F ) following laser irradiation. Hatched and solid black columns show the data at times 24 and 48 h after lesioning, respectively. The data are represented as the mean + S.E.M. (n = 21-25). Asterisks indicate significant differences from the values in group which received axonal lesions but was not treated with polyamines (Cont): * *P < 0.01; Dunnett's test.
of neurons. The cells were photographed immediately after laser irradiation and then drugs were added to the culture medium. The same cells were photographed 24 and 48 h after addition of drugs. If the selected neurons had died within 48 h, the data of the cells were omitted. Measurement of morphological parameters was made by tracing the photographs on a digitizing tablet. Possible responses of neurons following axonal injury are (1) neurite regeneration from injured site, (2) changes in axonal branches at proximal part of injured
axon, and (3) changes in uninjured dendrites. Furthermore, neuritic shape is formed by two different processes, 'elongation' and 'branching'. Therefore, we measured the following morphological parameters: (1) axon length from soma to injured site, (2) regenerated axon length from injured site, (3) the number of branch points in regenerated axon, (4) total length of regenerated axon including branches, (5) the number of branch points in proximal part of injured axon, (6) total length of branches in proximal part of injured axon, (7) the number of primary neurites per soma, and 8) total
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0
25
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Fig. 5. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on the number of branch points ( A - C ) and total length of branches ( D - F ) in proximal part of injured axons. White, hatched and solid black columns show the data at times 0, 24 and 48 h after lesioning, respectively. The data are represented as the mean + S.E.M. (n = 21-25).
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
from soma (Fig. 6A) and total length of uninjured dendrites (Fig. 6D) were not affected by the addition of spermine. The axonal regeneration-promoting effect of spermine was concentration dependent and seen maximally at 10 -8 M. Spermidine and putrescine also promoted the re-elongation of injured axons with the maximum effect at a concentration of 10 -8 M (Fig. 4B and C). The growth of branches in proximal part of injured axons and uninjured dendrites were not affected by spermidine or putrescine (Fig. 5B,C,E,F and Fig. 6B,C,E,F). The effects of three polyamines were very similar in terms of both the effective concentrations and the patterns of morphological changes induced by them, implying that all three polyamines may act through a common mechanism. To examine this possibility, we investigated whether or not the additivity was seen when polyamines were added together. As shown in Fig. 7A, axonal re-elongation induced by spermine, spermidine and putrescine at a maximally effective concentration (10 -s M) were virtually the same, and no additivity was seen when spermine was added together with spermidine or putrescine. Concomitant addition of spermine and spermidine or putrescine affected neither the branches in proximal part of injured axons (Fig. 7B) nor the uninjured dendrites (Fig. 7C and D). Furthermore, the effect of spermine was compared with that of bFGF. Addition of b F G F (1 ng/ml), unlike spermine, did not promote the re-elongation of injured axons (Fig. 8B and C), but the number of branch points and total length of branches in proximal part of injured axons were dramatically increased by b F G F (Fig. 8E and F). The numbers of primary neu-
length of uninjured dendrites. Definition of each morphological parameter is illustrated in detail in Fig. 1.
3. Results
First we checked the influence of laser irradiation on morphology of cultured hippocampal neurons. Axonal elongation ceased following the laser irradiation, but the growth of branches at proximal part of axon, the number of neurites per soma and dendritic growth were not affected by the laser irradiation (Figs. 2 and 3A). Next the effects of polyamines on the morphological changes following axonal lesioning were investigated. Since the distance of the injured site from the soma may influence the regenerative response of neurons [10], we confirmed that the axon length from soma to the injured site was not significantly different among the tested groups (data not shown). Fig. 3B shows micrographs of a representative neuron cultured in the medium with added spermine. The results of quantitative analyses in many neurons are shown in Figs. 4-6. Addition of spermine greatly promoted the re-elongation of axons from the injured site (Fig. 4A). No significant increase in branch points in regenerated axons (Fig. 4D) means that injured axons extended without bifurcating. Spermine did not affect the number of branch points in proximal part of injured axons (Fig. 5A). Total length of branches in proximal part of injured axon tended to be increased by the presence of spermine, but the effect was not statistically significant (Fig. 5D). The number of primary neurites emanating
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e
7
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Fig. 6. The effects of spermine (A,D), spermidine (B,E) and putrescine (C,F) on the n u m b e r of primary neurites per soma ( A - C ) and the total length of uninjured dendrites ( D - F ) . White, hatched and solid columns show the data at times 0, 24 and 48 h after lesioning, respectively. The data are represented as the m e a n +. S.E.M. (n = 21-25).
238
P.-j. Chu et aL / B r a i n Research 673 (1995) 233-241
rites per soma (Fig. 8G) and total length of uninjured dendrites (Fig. 8H) were not affected by bFGF. When spermine (10 -8 M) and b F G F (1 n g / m l ) were added together, both the axonal re-elongation from injured site and the growth of branches in proximal part of injured axons were promoted (Fig. 8B,C and F). Interestingly, the number of branch points in regenerated axons, which was not significantly affected by spermine or b F G F alone, was significantly increased by concomitant addition of spermine and b F G F (Fig. 8D). The number of primary neurites per soma and the growth of uninjured neurites were not altered by simultaneous addition of spermine and b F G F (Fig. 8G and H). The effects of spermine and b F G F are schematically summarized in Fig. 9.
Therefore, it is probable that three polyamines act through a common mechanism. Polyamines are known to potentiate the N-methyl-D-aspartate (NMDA) receptor-mediated responses [7,18,21,26,29]. However, Mattson et al. [14] have reported that neurite outgrowth of cultured hippocampal neurons was reduced
100
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4. D i s c u s s i o n
The main finding in the present study is that spermine, spermidine and putrescine were very effective in promoting the re-elongation of injured axons of cultured rat hippocampal neurons. First, the following data support that the effects of polyamines observed in the present study is not due to a secondary effect of enhanced survival of neurons: (1) the moderate condition of laser irradiation employed in the present study stopped the axonal growth, but did not affect the survival and other morphological parameters of neurons; (2) we have previously observed that spermine was effective in supporting the survival of cultured rat hippocampal neurons but spermidine and putrescine had no effect [1,6], whereas, in the present study, three polyamines similarly promoted the axonal re-elongation. Furthermore, the re-elongation of injured axons of neurons which once grew and differentiated in culture seems to be, at least in part, different from the initial phase of growth of dissociated neurons in monolayer culture. For example, interleukin-2 promoted axonal elongation of dissociated hippocampal neurons in normal culture condition [25], but did not promote re-elongation of axons injured by laser irradiation [our unpublished observation]. On the other hand, polyamines more effectively promoted the re-elongation of injured axons than the axonal elongation in normal culture condition [6]. Therefore, the present result suggests that polyamines have a capability of promoting axonal regeneration of brain neurons after lesioning. The mechanism by which polyamines promote the axonal re-elongation is not yet clear. The effects of spermine, spermidine and putrescine were very similar in terms of both the mode of action and effective concentrations, and the concomitant addition of spermine and spermidine or putrescine at a maximally effective concentration showed no additivie effect.
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Fig. 7. No additivity of the effects of spermine (SPN), spermidine (SPD) and putrescine (PUT) on axonal regeneration followinglesioning. Polyamines at a maximally effective concentration (10 s M) were added alone or concomitantly, and the effects on regenerated axon length from injured site (A), the number of branch points in proximal part of injured axons (B), the number of primary neurites per soma (C) and the total length of uninjured dendrites (D) were compared. The date are represented as mean _+S.E.M. (n = 20-24). Asterisks indicate significant differences from the value of control group: * *P < 0.01; Dunnett's test.
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
239
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Fig. 8. Comparison of the effects of spermine (SPN) and b F G F on neuronal morphology following axonal lesioning. Spermine (10 -8 M) and b F G F (1 n g / m l ) were added alone or concomitantly immediately after laser irradiation, and the morphological parameters were measured at times 0 (white columns), 24 (hatched columns) and 48 h (solid black columns) after addition of drugs. The data are represented as the m e a n + S.E.M. (n = 21-23). Asterisks indicate significant differences from the value of control group: * *P < 0.01; D u n n e t t ' s test.
v
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+ Spermine / bFGF
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Fig. 9. Summary of the effects of spermine and bFGF on neurite regeneratoin following axonal lesioning. Spermine selectively promoted the axonal re-elongation from injured site, while bFGF predominantly stimulated the formation of axonal branches at proximal part of injured axons. Uninjured dendrites were affected by neither spermine nor bFGF. Concomitant addition of spermine and bFGF promoted both the axonal re-elongation from injured site and the axonal branch formation at proximal part of injured axons. Furthermore, branching of regenerated axons occurred by synergistic action of spermine and bFGF.
by kainate and quisqualate but not by N M D A , suggesting that N M D A receptors are not involved in regulating neurite outgrowth. Moreover, putrescine is virtually inactive in potentiating the activation of N M D A receptor channel complex [7,23,24], but promoted the axonal re-elongation as well as spermine and spermidine. Therefore, it is unlikely that polyamines promote the axonal re-elongation by potentiating N M D A response. Alternatively, it is known that polyamines interact with D N A or R N A and stimulate protein biosynthesis in various types of cells [27]. Therefore, it is possible that polyamines regulate the protein synthesis associated with the neuronal regeneration. Polyamines did not affect the growth of branches in proximal part of injured axons and uninjured dendrites. The reason why polyamines promotes predominantly the axonal reelongation of injured axons is not clear, but it is possible that polyamines selectively regulate some cytoskelton component associated with axonal regeneration. In addition, it is also possible that the polyamines affect Ca 2+ homeostasis in hippocampal neurons, for Ca 2+ plays a key role in neurite outgrowth and regeneration
240
P.-j. Chu et al. /Brain Research 673 (1995) 233-241
[12,15]. F u r t h e r investigations a b o u t the m e c h a n i s m of action of the p o l y a m i n e s are b e i n g c o n d u c t e d in o u r laboratory. T h e specificity of the action of p o l y a m i n e s was conspicuous by a c o m p a r i s o n with the effect of b F G F . It has b e e n r e p o r t e d that b F G F p r o m o t e d the survival [2,17,28] a n d n e u r i t e growth of c u l t u r e d b r a i n n e u r o n s [3,15] a n d that b F G F increases in n e u r a l tissues of chemically or m e c h a n i c a l l y lesioned b r a i n [9,13,22], suggesting that b F G F may be also involved in the n e u r o n a l r e g e n e r a t i o n in pathological conditions. U n like polyamines, b F G F had n o capability to p r o m o t e the axonal r e - e l o n g a t i o n from i n j u r e d site, s u p p o r t i n g that the p r o m o t i o n of the axonal r e - e l o n g a t i o n is a specific action of polyamines. Instead, b F G F stimulated the b r a n c h f o r m a t i o n at proximal part of i n j u r e d axons, c o n s i s t e n t with o u r previous observation [16]. S p e r m i n e a n d b F G F seem to rescue the i n j u r e d n e u r o n s t h r o u g h different strategies: s p e r m i n e promotes axonal r e - e l o n g a t i o n from i n j u r e d site, while b F G F stimulates the f o r m a t i o n of axonal b r a n c h e s at proximal part of i n j u r e d axon. W h e n s p e r m i n e a n d b F G F were a d d e d together, both axonal r e - e l o n g a t i o n a n d b r a n c h i n g were p r o m o t e d , suggesting that axonal r e - e l o n g a t i o n and b r a n c h i n g are i n d e p e n d e n t l y regulated by different factors or m e c h a n i s m s . Interestingly, the n u m b e r of b r a n c h points in r e g e n e r a t e d axons was significantly increased by synergistic action of s p e r m i n e a n d b F G F . This effect a p p e a r s to result from that axons r e g e n e r a t e d by the action of s p e r m i n e were f u r t h e r s t i m u l a t e d to bifurcate by b F G F . T h e s e data d e m o n s t r a t e that the c o m b i n e d use of m o r e t h a n o n e drugs that act t h r o u g h different m e c h a n i s m s has the a d v a n t a g e of m o r e effectively p r o m o t i n g the recovery following n e u r o n a l injury. In conclusion, we have shown for the first time that s p e r m i n e , s p e r m i d i n e a n d p u t r e s c i n e strongly a n d selectively p r o m o t e the r e - e l o n g a t i o n of axons of cult u r e d h i p p o c a m p a l n e u r o n s following axonal lesions. It is possible that p o l y a m i n e s play a role in n e u r o n a l r e g e n e r a t i o n following b r a i n injury. I n addition, conc o m i t a n t a d d i t i o n of s p e r m i n e a n d b F G F additively or synergistically p r o m o t e d the axonal re-growth from inj u r e d site a n d the b r a n c h f o r m a t i o n at proximal part of i n j u r e d axons, suggesting the a d v a n t a g e of c o m b i n e d use of m o r e t h a n o n e drugs for t h e r a p y of n e u r o d e g e n erative disorders.
Acknowledgements T h e a u t h o r s are grateful to T a k e d a C h e m i c a l I n d u s tries, Ltd. for the g e n e r o u s gift of b F G F .
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