Brain Research 970 (2003) 250–253 www.elsevier.com / locate / brainres
Immunosuppressive (FK506) and non-immunosuppressive (GPI1046) immunophilin ligands activate neurotrophic factors in the mouse brain Ken-ichi Tanaka*, Naoko Fujita, Norio Ogawa Department of Brain Science, Okayama University Graduate School of Medicine and Dentistry, 2 -5 -1 Shikatacho, Okayama 700 -8558, Japan Accepted 4 February 2003
Abstract Based on the fact that several recent reports have indicated that non-immunosuppressive immunophilin ligands (IPLs) can activate neurite outgrowth or nerve regeneration, we investigated the neurotrophic factor-activating abilities of IPLs in vivo in order to clarify the molecular basis of neurotrophic-like activity. Both FK506 (an immunosuppressive IPL) and GPI1046 (a non-immunosuppressive IPL) significantly increased glial cell line-derived neurotrophic factor (GDNF) content in the substantia nigra. In addition, FK506 increased striatal brain-derived neurotrophic factor (BDNF) content significantly. Thus, our present results suggest that the molecular basis of IPL-induced neurotrophic-like activity may be dependent on GDNF and / or BDNF activation. 2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Non-immunosuppressive immunophilin ligand; Glial cell line-derived neurotrophic factor; Brain-derived neurotrophic factor; GDNF family receptor a-1; Trk B
Non-immunosuppressive immunophilin ligands (IPLs) are attracting attention as new candidate drugs for neuroprotection and neurorestoration, particularly because they do not have the adverse effects of immunosuppressants [5,6,9,15–17]. For example, it has been shown that noncalcineurin binding analogues of FK506, such as GPI1046 and V10367, which are not immunosuppressive, still promote neurite outgrowth [6,16] and prevent cell death [18,20], although several researchers have postulated that non-immunosuppressive IPLs do not have any beneficial effects [8,22]. We previously demonstrated that GPI1046, by increasing intracellular glutathione (GSH) levels in several cell lines, had neuroprotective effects equal to those of FK506 against hydrogen peroxide-induced apoptosis [18,20,21]. In an in vivo study, FK506 (0.5 mg / kg per day) or GPI1046 (10 mg / kg per day) was found to prevent the reduction of striatal dopamine (DA) concentrations, and normalized completely the acceleration of *Corresponding author. Tel.: 181-86-235-7410; fax: 181-86-2357412. E-mail address: [email protected]
striatal DA turnover induced by 6-hydroxydopamine (6OHDA) lesions . Both FK506 and GPI1046 were found to increase striatal GSH contents as a result of activating GSH-related enzyme mRNA . Therefore, we concluded that GPI1046 may have neuroprotective effects both in cell cultures and in vivo. In addition, several recent reports have indicated that non-immunosuppressive IPLs have neurotrophic-like activity such as the activation of neurite outgrowth and / or nerve regeneration [6,16]. However, the molecular basis of neurotrophic-like activity of IPLs remains obscure. Accordingly, the present study was conducted to confirm the molecular basis of neurotrophiclike activities of IPLs in vivo. Neurotrophic factors (NTFs) are essential for the survival and differentiation of normally developing neurons, but they also play important roles in the protection and recovery of mature neurons under pathologic conditions [1,12]. However, in vivo treatment of NTFs as neuroprotective agents faces a serious problem: NTFs have a low degree of penetration of the blood–brain barrier (BBB). Therefore, a low molecular weight compound mimics and / or enhances the neurite outgrowth and regene-
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02434-X
K.-i. Tanaka et al. / Brain Research 970 (2003) 250–253
ration. Many NTFs have been shown to promote DArgic neuronal systems both in cell cultures and in vivo. In particular, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) among the NTFs show potent selectivity in promoting the survival and / or the protection of DArgic neurons from neurotoxicity induced by DArgic toxins such as 6-OHDA [4,7,10]. Thus, low molecular weight compounds that possess activating properties of GDNF and / or BDNF may be useful as therapeutic drugs for Parkinson’s disease (PD). Male ICR mice (Charles River, Yokohama, Japan) weighing 28–30 g were provided with free access to food and water while housed under a 12-h light / 12-h dark cycle (lights on at 07:00 h) at constant room temperature (2461 8C) and humidity (55%) for 1 week before the experiments. Animal care and all experimental procedures were in strict accordance with the Guidelines for Animal Experiments of Okayama University Graduate School of Medicine and Dentistry. In all experiments, FK506 (placebo-vehicle or 0.5 mg / kg; Fujisawa Pharmaceutical, Osaka, Japan), or GPI1046 (1.0% dimethyl sulfoxide (DMSO) or 10 mg / kg) was administered subcutaneously (s.c.) for 7 consecutive days. A final concentration of DMSO of #1.0% was maintained. After sacrifice by decapitation, the striatum and midbrain including substantia nigra tissues were rapidly preserved at 280 8C until analysis. GDNF or BDNF levels were determined by sandwich ELISA using a commercially available kit according to the manufacturer’s instructions (GDNF Emax姠 Immunoassay System, BDNF Emax姠 Immunoassay System; Promega, Madison, WI). In addition, expression levels of the GDNF family receptor a-1 (GFRa-1) or Trk B were examined by Western blot analysis. Blots were incubated with rabbit anti-mouse GFRa-1 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:250 dilution) or rabbit anti-mouse Trk B (Santa Cruz Biotechnology, Santa Cruz, CA; 1:250 dilu-
tion) polyclonal antibody, then reacted with goat antirabbit secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA). After a washing with 20 mM Tris-buffered saline containing 0.1% Tween 20, blots were developed using the ECL Western blotting detection system (Amersham, Buckinghamshire, UK) according to the protocol provided by the manufacturer. For quantitative analysis, sample loading and transfer were normalized by using goat anti-human / mouse actin polyclonal antibody (Santa Cruz Biotechnology; 1:250 dilution). The protein concentration of brain homogenates was determined using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA) in order to revise the NTF content. Data are presented as the means6S.E.M. Differences between groups were tested for statistical significance using oneway analysis of variance (ANOVA) followed by post hoc Duncan’s multiple range test. A P-value ,0.05 denoted the presence of a statistically significant difference. The substantia nigral GDNF level was significantly increased by administration of FK506 or GPI1046, though the striatal GDNF level was not changed by administration of FK506 or GPI1046 (Table 1). The substantia nigral GFRa-1 expression was significantly increased by administration of FK506 or GPI1046, though the striatal GFRa-1 expression was not changed by administration of FK506 or GPI1046 (Table 1). GDNF binds first to the glycosylphosphatidylinositol-anchored GFRa-1, then the GDNF–GFRa-1 complex binds to, and stimulates autophosphorylation of, Ret, which is a transmembrane receptor tyrosine kinase. However, recent reports have suggested that GFRa-1 evoked GDNF-induced intracellular signaling in the absence of Ret . At present, the molecular basis of GDNF’s action is far from clear, despite the potential clinical importance of GDNF . The striatal BDNF level was significantly increased by administration of FK506, but not by GPI1046, although the substantia nigral BDNF level was not changed by administration of FK506 or GPI1046 (Table 2). The striatal Trk B
Table 1 Effects of FK506 or GPI1046 on GDNF concentration and GFRa-1 expression in mice FK506
0.5 mg / kg per day
10 mg / kg per day
GDNF concentration ( pg /mg protein) Striatum 8.39560.209 Substantia nigra 2.21860.281
GFRa -1 expression (ratio of GFRa -1 /Actin) Striatum 1.08260.035 Substantia nigra 1.06860.039
Each entry represents the mean6S.E.M. (n56–10). *P,0.05, compared with vehicle-treated mice. **P,0.01 compared with vehicle-treated mice.
K.-i. Tanaka et al. / Brain Research 970 (2003) 250–253
Table 2 Effects of FK506 or GPI1046 on BDNF concentration and Trk B expression in mice FK506
0.5 mg / kg per day
10 mg / kg per day
BDNF concentration ( pg /mg protein) Striatum 1.42360.310 Substantia nigra 4.22960.341
Trk B expression (ratio of Trk B /actin) Striatum 1.25760.023 Substantia nigra 0.97160.009 Each entry represents the mean6S.E.M. (n56–10). *P,0.05, compared with vehicle-treated mice. **P,0.01 compared with vehicle-treated mice.
expression was significantly decreased by administration of FK506, but not by GPI1046, although the substantia nigral Trk B expression was not changed by administration of FK506 or GPI1046 (Table 2). BDNF binds to and activates the tyrosine kinase receptor Trk B, stimulates regeneration and sprouting of surviving nigral neurons , and increases the activity of DA neurons  by initiating intracellular signaling [3,23]. Enhanced BDNF signaling may trigger cellular protective responses, such as preservation of calcium homeostasis, and prevention of energetic metabolism failure [2,11]. As a consequence, the target molecules of IPLs may be mainly NTFs, but not NTF’s receptor proteins. Because both GFRa-1 and Trk B expression levels were decreased slightly by repeated treatment with IPLs, IPL-induced reduction of GFRa-1 and Trk B expression may thus be only compensatory changes. In addition, we investigated whether FK506 and GPI1046 also had the same activating effect as GDNF and / or BDNF in 6-OHDA-lesioned mice (unpublished observations). In conclusion, our present results suggest that the molecular basis of IPL-induced neurotrophic-like activity may be dependent on GDNF and / or BDNF activation, although IPL-induced activation of NTFs may be dependent on the increased number of surviving cells. Furthermore, IPLs may enhance and / or mimic the NTFs’ functions, such as promotion of neuronal survival or prevention of neuronal degeneration, since the BBB is more permeable to IPLs than to NTFs. Thus, IPLs may be more useful than NTFs as therapeutic drugs against PD progression.
Acknowledgements This work was supported in part by Grants-in-Aid for Comprehensive Research on Aging and Health, and for Research on Brain Science from the Japanese Ministry of Health, Labour and Welfare, and Grants-in-Aid for Sci-
entific Research (C), and for Encouragement of Young Scientists from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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