Stimulation of nicotinic acetylcholine receptors protects motor neurons

BBRC Biochemical and Biophysical Research Communications 330 (2005) 1285–1289 www.elsevier.com/locate/ybbrc

Stimulation of nicotinic acetylcholine receptors protects motor neurons Tomoki Nakamizo a, Jun Kawamata b, Hirofumi Yamashita a, Rie Kanki a, Takeshi Kihara c, Hideyuki Sawada a, Akinori Akaike c, Shun Shimohama a,* a b

Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan Horizontal Medical Research Organization, Kyoto University Faculty of Medicine, Yoshida konoe-cho, Sakyoku, Kyoto 606-8501, Japan c Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi-cho, Sakyoku, Kyoto 606-8501, Japan Received 18 March 2005 Available online 25 March 2005

Abstract The present study demonstrated that administration of nicotine prevented glutamate-induced motor neuronal death in primary cultures of the rat spinal cord. The nicotine-induced neuroprotection was inhibited by either dihydro-b-erythroidin (DHbE) or a-bungarotoxin (aBT), suggesting that it is mediated through both a4b2 and a7 nicotinic acetylcholine receptors (nAChRs). Both a4b2 and a7 nAChRs were identified on rat spinal motor neurons by immunohistochemical methods. We also demonstrated that galantamine, an acetylcholinesterase inhibitor with allosteric nAChR-potentiating ligand properties, prevented glutamate-induced motor neuronal death. These results suggest that stimulation of nAChR may be used as a treatment for ALS.
2005 Elsevier Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Nicotine; Nicotinic acetylcholine receptor; Acetylcholinesterase inhibitor; Galantamine; Spinal cord; Motor neuron

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the rapidly progressive degeneration of motor neurons resulting in paralysis and, within a few years, death. In some types of familial ALS (FALS), mutations of the superoxide dismutase 1 (SOD1) gene have been demonstrated [1]. However, the cause of sporadic ALS is still unknown and with the exception of the recently introduced glutamate-release inhibitor, riluzole [2], no effective treatments have been identified. Therefore, it is important to develop a treatment for ALS. Although the pathogenesis of sporadic ALS is unclear, several clinical studies suggest the role of glutamate-induced excitotoxicity [2– 4]. Our previous study demonstrated that rat spinal cord cultures exposed to long-term (24 h) low-dose glutamate *

Corresponding author. Fax: +81 75 751 3265. E-mail address: [email protected] (S. Shimohama). 0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.115

exhibit selective motor neuronal death and proposed this paradigm as an in vitro model for ALS [5]. It has been reported that the stimulation of nicotinic acetylcholine receptors (nAChR) affords neuroprotection against cerebral ischemia [6] and ParkinsonÕs disease [7] in vivo. In vitro studies have also demonstrated that nicotine protects cerebral neurons against excitotoxicity [8–10], b-amyloid toxicity [11,12], and 1-methyl-4-phenylpyridinium-induced toxicity [13]. Acetylcholinesterase inhibitors (AChE-Is), which stimulate cholinergic transmission, are used for the treatment of AlzheimerÕs disease. Also, there is a study which demonstrates an early decrease in cholinergic input on motor neurons in the spinal cords of patients with ALS [14]. In the present study, we investigated the neuroprotective effect of nicotine and galantamine, an AChE-I with allosteric nAChR-potentiating properties, against spinal motor neuronal death induced by glutamate using dissociated cultures of fetal rat spinal cord.

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Materials and methods Experiments were carried out on animals treated in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Materials. Nicotine was purchased from Nacalai Tesque (Kyoto, Japan). Dihydro-b-erythroidin (DHbE) and a-bungarotoxin (aBT) were purchased from Sigma (St. Louis, MO). Galantamine was purchased from Tocris (Bristol, UK). The anti-a4 nAChR and anti-a7 nAChR antibodies were purchased from Sigma. The monoclonal antibody SMI32 was purchased from Sternberger Monoclonals (Baltimore, MD). L-trans-Pyrrolidine-2,4-decarboxylate (PDC) was purchased from Tocris. Spinal cord cultures. Dissociated cell cultures of the ventral half of the spinal cord removed from fetal Wistar rats on day 16 of gestation were prepared according to methods described previously [5]. Treatment of culture. Cultures were exposed to a medium containing 10 lM L-glutamate and 20 lM L-trans-pyrrolidine-2,4-decarboxylate (PDC), a glutamate transporter inhibitor, for 24 h on day 11 in vitro. The cultures were then incubated for another 48 h and fixed on day 14 for immunocytochemical evaluation. Control experiments were given identical treatment but using medium containing no drugs. The neuroprotective effect of nicotine or galantamine was evaluated by adding them to the medium in addition to glutamate. Evaluation of neurotoxicity. Neurotoxicity was assessed by counting the number of viable neurons after immunostaining with SMI32 antibodies. It has been demonstrated that large (>20 lm) neurons stained with SMI32 represent motor neurons in the spinal cord [5]. Immunostaining was performed using previously described methods [5]. The number of large neurons stained with SMI32 in 25 randomly selected fields (200·) was taken as the number of surviving motor neurons. Neurotoxicity in each treatment was assessed as the difference between the mean density (cells/mm2) of viable motor neurons in

cultures exposed to neurotoxins and in control cultures, and expressed as the reduction rate compared with control cultures (% of control). Counting was performed blind to the treatment each culture dish had received. Statistical analysis was performed by one-way analysis of variance and post hoc multiple comparison using Bonferroni/DunnÕs method. Uniformity of variance was tested using BarlettÕs test. Statistical significance was defined as p < 0.05. Immunohistochemistry of nAChRs. An 8-week-old male Wistar rat was deeply anesthetized with diethyl ether and perfused transcardially with cold phosphate-buffered saline (PBS) followed by 4% PFA solution. The lumbar spinal cord was excised and postfixed overnight with 4% PFA. The tissue was then cryoprotected in increasing concentrations of 15%, 20%, and 25% sucrose solutions. Ten micrometer thick frozen sections were cut in a cryostat and mounted on slides. After treating with 0.3% hydrogen peroxide in methanol for 20 min and blocking with 5% normal goat serum in PBS for 2 h at room temperature, the sections were incubated with either the anti-a4 nAChR or anti-a7 nAChR primary antibody overnight at 4 C. The sections were then incubated with biotinylated goat anti-rabbit immunoglobulin G overnight at 4 C followed by incubation with the avidin–biotin complex solution for 1 h at room temperature. Finally, the sections were incubated with a solution of diaminobenzidine solution for 2 min.

Results Neuroprotection provided by nicotine and galantamine against chronic glutamate-induced neurotoxicity Twenty-four hour exposure to low-dose (10 lM) glutamate with PDC (20 lM) caused a significant reduction

Fig. 1. (A) Protection of spinal motor neurons against chronic, low-dose glutamate (Glu)-induced toxicity by nicotine. Twenty-four hour exposure to 10 lM Glu with 20 lM PDC injured motor neurons. Nicotine significantly reduced motor neuronal death in a dose-dependent manner. *p < 0.05, ** p < 0.01 Glu vs. Glu + nicotine. (B) The neuroprotection provided by nicotine was blocked by 30 nM DHbE or 1 nM aBT, an a4b2 nAChR or a7 nAChR antagonist, respectively. **p < 0.01 vs. Glu, ##p < 0.01 vs. Glu + nicotine. Each datum represents the mean ± SEM of four independent experiments.

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Fig. 2. Protection of spinal motor neurons by galantamine. Twentyfour hour exposure to 10 lM Glu with 20 lM PDC injured motor neurons. Galantamine significantly reduced motor neuronal death in a dose-dependent manner. **p < 0.01 vs. Glu. Each datum represents the mean ± SEM of eight independent experiments.

in the number of surviving motor neurons in vitro. Administration of nicotine significantly attenuated the neurotoxicity in a dose-dependent manner (Fig. 1A). The protective effect of nicotine was significantly blocked by 30nM DHbE, an a4b2 nAChR antagonist, or 1 nM aBT, an a7 nAChR antagonist (Fig. 1B). Treatment with the same concentration of these antagonists alone did not affect glutamate-induced neurotoxicity (data not shown). Administration of galantamine also significantly attenuated the neurotoxicity against motor neurons dose-dependently. (Fig. 2). Immunohistochemistry of nAChRs Staining of both a4 and a7 subunits of nAChR was strongly positive on the soma of the motor neurons in the rat spinal cord. (Fig. 3). Also, a4 nAChRs were located on several neurons in the pericanal gray matter and a7 nAChRs were located on many of the dorsal neurons (data not shown).

Discussion It has been reported that nicotine exerts neuroprotective effects on neurons and many reports have demon-

Fig. 3. Immunostaining of a4b2 nAChR (A) and a7 nAChR (B) on the anterior horn of the rat lumbar spinal cord. Motor neurons are immunoreactive with both a4b2 nAChR and a7 nAChR. Bars represent 100 lm.

strated the involvement of nAChRs. Although 16 mammalian genes encoding for subunits of these receptors have been identified, the two main subtypes of nAChR in the central nervous system are a4b2 and a7 AChRs. Both a4b2 [7,8,12] and a7 [9–11,15,16] subtypes have been implicated in the mechanism of neuroprotection provided by nicotine. In this study, we demonstrated that the neuroprotection elicited by nicotine was inhibited by both a4b2 and a7 nAChR antagonists, suggesting that both subtypes were involved. Consistent with this finding was the demonstration of both a4b2 and a7 nAChRs on spinal motor neurons by immunohistochemistry. Until now, there has been only one study showing that nicotine exerted neuroprotection on spinal motor neurons [15]. Using pure embryonic motor neuronal cultures, that study demonstrated that nicotine prevented naturally occurring apoptosis and that the neuroprotection was blocked by an a7 nAChR antagonist but not by an a4b2 nAChR antagonist. In the present study, the

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neuroprotection elicited by nicotine was blocked by both a7 nAChR and a4b2 nAChR antagonists. The cause of this apparent discrepancy is not clear, but there are a couple of possible explanations. First, naturally occurring embryonic motor neuron death may be an event specific to that stage. In our cultures, however, neurons had been cultured for 11 days when the toxic stimuli were applied. Second, the difference in cultural composition (pure motor neuron vs. mixed neuron-glial culture) might be a contributing factor. We have previously demonstrated the involvement of activation of the PI3-kinase-Akt system and up-regulation of Bcl-2 in nAChR-mediated neuroprotection using cultured cortical neurons [16]. Aberration in the PI3-kinase-Akt signaling system has been reported both in both ALS patients [17] and ALS transgenic mice [18]. Thus, it is possible that the PI3-kinase-Akt cascade is involved in nAChR-mediated neuroprotection against glutamate-induced spinal motor neuronal death. Galantamine is an AChE-I that is currently used for the treatment of AlzheimerÕs disease. In addition to the inhibition of AChE, galantamine binds to nAChRs and allosterically potentiates their synaptic transmission [19]. Consequently, galantamine is called an allostericpotentiating ligand (APL) of nAChRs. This APL effect is present on both a7 and a4b2 nAChRs [19]. Thus, galantamine could stimulate cholinergic transmission in two ways: (1) by inhibiting AChE and increasing AChs and (2) by potentiating cholinergic transmission through the APL effect. Recently, we demonstrated that galantamine protected cortical neurons against Ab-enhanced glutamate toxicity by, at least partially, the a7 nAChR-PI3-kinase-Akt cascade [20]. In the present study, galantamine has a neuroprotective effect on motor neurons. This result suggests that galantamine, and possibly other AChE-Is, could be used for the treatment of ALS. There have been no reports on the use of galantamine for the treatment of ALS. Galantamine is widely used as a treatment for AlzheimerÕs disease and, therefore, its safety has been well proven. Accordingly, it might be a good candidate for the treatment of ALS. In conclusion, the results of this study raise the possibility that cholinergic stimulation by AChE-Is, such as galantamine or by nAChR agonists, could be used as a treatment for ALS, although further studies are necessary to elucidate the neuroprotective effects of nAChR stimulation on motor neurons.

Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science, Research Grant on Measures for Intractable Disease from

the Ministry of Health, Labor and Welfare of Japan, and grants from the Smoking Research Foundation, Philip Morris USA Inc., and Philip Morris International.

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