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Galanin up-regulates the expression of M1 muscarinic acetylcholine receptor via the ERK signaling pathway in primary cultured prefrontal cortical neurons
Neuroscience Letters 590 (2015) 161–165
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Galanin up-regulates the expression of M1 muscarinic acetylcholine receptor via the ERK signaling pathway in primary cultured prefrontal cortical neurons Yong Cheng, Long-Chuan Yu ∗ Laboratory of Neurobiology and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China
h i g h l i g h t s • Galanin up-regulates the expression of M1 receptor in the neurons. • The ERK signaling pathway mediates the galanin induced up-regulation of M1 receptor. • Our study supports the hypothesis that galanin is neurotrophic in the progress of AD.
a r t i c l e
i n f o
Article history: Received 21 November 2014 Received in revised form 3 February 2015 Accepted 6 February 2015 Available online 9 February 2015 Key words: Galanin M1 muscarinic acetylcholine receptor Alzheimer’s disease ERK
a b s t r a c t The expression of galanin and galanin receptors are up-regulated in the brains from patients with Alzheimer’s disease (AD). However, the role of galanin in the progress of AD is still controversial. Here we demonstrated that galanin increased the protein expression of M1 muscarinic acetylcholine receptor (M1) in the primary cultured prefrontal cortical neurons by ELISA and Western Blot. Moreover, we showed that the mRNA expression of M1 was also up-regulated by galanin treatment. We further explored the mechanism of the galanin induced up-regulation of M1. We found that galanin activated the ERK signaling pathway in the primary cultured prefrontal cortical neurons. In addition, our results showed that the up-regulation of M1 mRNA was blocked by an ERK inhibitor, U0126. Taken together, our results demonstrated that the ERK signaling pathway mediated the galanin induced up-regulation of M1 in the primary cultured prefrontal cortical neurons, supporting the hypothesis that galanin plays a beneficial role in the development of AD. © 2015 Elsevier Ireland Ltd. All rights reserved.
Galanin is a 29 amino acids neuropeptide with wide expression in the central and peripheral nervous systems [18]. Galanin has been reported to be involved in many biological functions such as nociception, feeding, waking and sleeping regulation, learning and memory [16,18]. Moreover, galanin is linked to a number of neurological disorders, including epilepsy, depression and Alzheimer’s disease (AD) [18]. Galanin had been found to be up-regulated in the post-mortem brains of patients with AD more than twenty years ago [1]. However, the role of galanin during the onset of AD is still poorly understood. AD is a progressive neurodegenerative disease with amyloid beta and tau protein as two major hallmarks [14]. Another major hallmark for AD is the hypofunction of cholingergic
∗ Corresponding author. Tel.: +86 10 62753667; fax: +86 10 62751526. E-mail address: [email protected] (L.-C. Yu). http://dx.doi.org/10.1016/j.neulet.2015.02.011 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
system in the brain. It has been reported that there are reduction of acetylcholine synthesis and acetylcholine receptor level as well as significant loss of cholinergic neurons in the brains from patients with AD [15]. The neurotransmitter acetylcholine activates either ionotropic nicotinic acetycholine receptors or metabotrophic muscarinic acetylcholine receptors (mAChRs) to have biological functions [15]. Five mAChR (M1–M5) subtypes have been identified to date and they are involved in various physiological functions in the nervous system. Of the five mAChRs, M1 plays a crucial role in neurological diseases including AD [17]. It has been shown that activation of M1 attenuated amyloid beta induced neurotoxicity through the WNT signaling pathway [13]. Moreover, deficiency in M1 was found to further exacerbate AD-like pathology in a mouse model of AD [22]. Interestingly, a study found that a selective M1 agonist reduced amyloid beta and tau pathology in a transgenic mice model of AD [2]. Thus M1 has been postulated as an important therapeutic target for treating AD.
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Fig. 2. Galanin increased the expression of M1 mRNA in the neurons. Top panel: Representative RT-PCR analysis of M1 mRNA expression after galanin treatment at different time points in the neurons. GAPDH served as internal control. Bottom panel: Bar graphs showing the quantification of the M1 mRNA signals normalized to GAPDH. Note galanin increased the M1 mRNA expression at a time depend manner. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control.
Fig. 1. Galanin up-regulated the M1 protein expression in the primary cultured prefrontal cortical neurons. (A) Galanin increased the surface M1 expression in the primary cultured prefrontal cortical neurons as tested by ELISA assay. Note galanin had the maximum effect on the expression of surface M1 at the concentration of 1 nM. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control. (B) Top panel: Representative Western Blot analysis of total M1 protein expression after galanin treatment at different time points in the neurons. Actin served as internal control. Bottom panel: Bar graphs showing the quantification of the total M1 protein signals normalized to actin. Note galanin increased the total M1 protein expression at a time dependent manner. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control. Four duplicates were run for the Western Blot.
Although the regulatory role of galanin on the release of acetylcholine had been reported by many groups [9,21,25], there is no study showing whether galanin regulates the acetycholine receptors in the brains. Here we demonstrated that galanin up-regulated the expression of M1 in the primary cultured prefrontal cortical neurons, both in protein and mRNA levels. Furthermore, we found that the ERK signaling pathway mediated the galanin induced upregulation of M1. The primary cultured prefrontal cortical neurons of rats were prepared as described previously [27]. Briefly, the dissociated neurons from postnatal day zero to one pups (mixed gender) were plated at 105 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 g/l HEPES, 2 g/l NaHCO3 , 100 U/mlstreptomycin and penicillin. To inhibit glial cell growth, cytosine arabinoside (10 M; Sigma, St.
Louis, Missouri) was supplemented after plating for 3 days. The neurons were used for experimentation after 7–8 days culture in vitro (DIV7-8). Galanin (Tocris, Bristol, UK) and 1,4-diamino-2,3dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126, Sigma, St. Louis, Missouri) were added freshly into the culture medium during treatments. U0126 is a highly selective inhibitor for extracellularsignal-regulated kinase 1/2 (ERK1/2). RT-PCRs were performed as described previously [20]. Briefly, primary cultured prefrontal cortical neurons were harvested and total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, California). Total RNA (2 ug) was reverse-transcribed using M-MLV Reverse Transcriptase (Invitrogen) and oligo-d(T) 15 random primers (Takara, Shiga, Japan). PCRs were done using Pre-mix Taq (Takara, Shiga, Japan). Sequences of the primers for the experiments were: rat M1: sense 5 -CAGTTCCTCTCCCAACCCAT3 and antisense 5 -TGGGCATCTTGATCACCACT-3 ; rat GAPDH: 5 -CGTATCGGACGCCTGGTT-3 and antisense 5 sense CCCTTCCACGATGCCAAAA-3 . Amplification was done at 94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min with a final extension cycle for 10 min at 72 ◦ C in an Eppendorf Master (Eppendorf, Hamburg, Germany). PCR products were electrophoresed on agarose gels, stained with ethidium bromide (Sigma, St. Louis, Missouri) and visualized under UV light. The results were assessed with a BioRad Chemi Doc XRS imaging system (BioRad Hercules, CA). Cell ELISA was used to detect M1 level on cell surface under non-permeabilized condition. The primary prefrontal cortical neurons of rats were treated and then gently washed with 0.01 M PBS before being fixed with 4% paraformaldehyde in PBS. Cells were then washed with PBS and blocked using PBS containing 10% donkey serum. After blocking, the cells were incubated with M1 primary antibody (1:200, Santa Cruz, CA, USA) diluted in 3% serum-PBS at 4 ◦ C overnight. Then cells were incubated with biotin-conjugated donkey anti-rabbit second antibody (1:1000) Jackson ImmunoResearch Laboratories, Inc., PA, USA) after washing with PBS, following by 0.3% H2 O2 incubation for 30 min to eliminate endogenous
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Fig. 3. The ERK1/2 signaling pathway mediated the galanin induced up-regulation of M1 in the neurons. (A) Top panel: Representative Western Blot analysis of p-ERK levels after 1 h or 3 h of galanin treatment in the primary cultured prefrontal cortical neurons. t-ERK served as internal control. Bottom panel: Bar graphs showing the quantification of p-ERK signals normalized to t-ERK 1/2. Note galanin activated the ERK signaling pathway in the neurons. Three duplicates were run for the Western Blot. (B) Top panel: Representative RT-PCR analysis of M1 mRNA expression after various treatments in the neurons. GAPDH served as internal control. Bottom panel: Bar graphs showing the quantification of M1 mRNA signals normalized to GAPDH. Note Galanin induced up-regulation of M1 mRNA expression was blocked by U0126 in the neurons, suggesting the ERK signaling pathway mediated the up-regulation of M1 induced by galanin. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control group; #p < 0.05 compared with galanin group.
peroxide enzyme. After washing with PBS, avidin–biotin complex (elite kit, Vector Laboratories, Burlingame, CA, USA) was added to the cells and incubated for 1 h. After washing, horseradish peroxidase (HRP) substrate 3,3 ,5,5 -tetramethylbenzidene (TMD) (0.1 mg/ml; Sigma, St. Louis, Missouri) was added to produce a color reaction that was stopped by 2N H2 SO4 . The optical density (absorbance) was read on a microplate reader (Model 550, Bio-Rad Hercules, CA) at 450 nm. Western Blot was performed as described previously [26,28]. Briefly, protein lysates of the primary cultured prefrontal cortical neurons were extracted after treatments. The lysates were iced for 30 min, centrifuged at 14,800×g for 15 min, and protein in the supernatant was harvested. The protein concentrations were determined using a spectrophotometric method. Denatured protein samples diluted with loading buffer were loaded equally into each lane, separated by 10% SDS-PAGE, and then blotted onto a polyvinylidene fluoride membrane. The membrane was then incubated for 1 h in blocking buffer (tris-buffered saline containing 5% defatted milk powder) at room temperature. Next, the membrane was incubated at 4 ◦ C with the primary antibodies, washed with tris-buffered saline and Tween-20 mixture and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by another wash. Immunoblots were developed in the presence of enhanced chemiluminescence reagents, and the images detected on X-ray film were quantified by densitometric scanning using a gel imaging analysis system. The purified polyclonal rabbit anti-t-ERK antibody (1:10000, Santa Cruz, CA, USA), polyclonal rabbit anti-M1 antibody (1:500, Santa Cruz, CA, USA), monoclonal mouse anti-p-ERK antibody (1:1000, Santa Cruz, CA, USA), polyclonal rabbit anti--actin antibody (1:3000, Santa Cruz, CA, USA),
HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, Inc., PA, USA), HRP-conjugated goat anti-mouse secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, Inc.), PVDF membrane (Millipore, MA, USA), X-ray film (Kodak, NY, USA) and Gel-Pro 4400 System (Media Cybernetics, Inc., MD, USA) were used in the experiments. Data were analyzed by one-way ANOVA followed by Tukey post-hoc multiple comparison test where noted. Significance was set at p < 0.05. To test whether galanin regulates the expression of M1, the primary cultured prefrontal cortical neurons were treated with vehicle, 100 pM, 1 nM,10 nM or 100 nM of galanin for 24 h, cell ELISA was then used to measure the expression level of M1 on cell membrane. As shown in Fig. 1A, galanin increased the expression of M1 on cell membrane at a dose dependent manner in the primary cultured prefrontal cortical neurons (ANOVA between control and treated groups, F(4,94) = 3.742, n = 20, p < 0.05), compared to the control group. Since the above results indicated that the effect of galanin on the expression of M1 peaks at 1 nM, we therefore used 1 nM of galanin in the subsequent experiments. We then used western blot to measure the total levels of M1 in primary cultured prefrontal cortical neurons. The neurons were treated with vehicle or 1 nM of galanin for 6 h, 12 h or 24 h and protein samples were collected for analysis. Results from western blot showed that the expression of total M1 was increased by galanin incubation (ANOVA between control and treated groups, F(3,15) = 5.819, n = 4, p < 0.05) in the neurons, compared to the control group, as shown in Fig. 1B. Our data have shown that galanin up-regulated the expression of M1 protein in the primary cultured prefrontal cortical
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neurons, we then tested mRNA expression of M1 after galanin treatment in the neurons. Results from RT-PCR demonstrated that the expression of M1 mRNA was increased after 6 h, 12 h, or 24 h of galanin treatment (ANOVA between control and treated groups, F(3,11) = 4.848, n = 4, p < 0.05) in the neurons, compared to the control group, as shown in Fig. 2. To elucidate the mechanism by which galanin mediated the synthesis of M1 in the neurons, we searched for the potential downstream signaling pathways that are known to be involved in the regulations of genes. We tested if galanin can activate the ERK signaling pathway, a signaling pathway that regulates gene expression and has been shown to be activated by galanin in hippocampus [12]. As showed in Fig. 3A, galanin increased the level of phosphorylated ERK after 1 h or 3 h treatment in the primary cultured prefrontal cortical neurons (ANOVA between control and treated groups, F(2,8) = 19.28, n = 3, p < 0.05), compared to the control group. However, the total ERK levels were not changed after galanin treatment, suggesting that galanin activated the ERK signaling pathway in the primary cultured prefrontal cortical neurons. To further investigate whether galanin induced up-regulation of M1 was mediated by the ERK signaling pathway in the neurons, we used the ERK inhibitor, U0126. Results from RT-PCR showed that pretreatment with 1 M U0126 for 30 min in the neurons completely blocked the increased expression of M1 mRNA induced by galanin (ANOVA between control and treated groups, F(2,8) = 9.67, n = 3, p < 0.05), as shown in Fig. 3B. The above results indicated that galanin induced up-regulation of M1 in the primary cultured prefrontal cortical neurons was mediated by the ERK signaling pathway. An important feature of AD is the increased expression of galanin and galanin receptors in the post-mortem brains of patients with AD [7]. Galanin was further found to be hyperinnervating the surviving cholinergic neurons in the brains from patients with AD [7]. Various studies showed that galanin had an inhibitory role in the release of acetycholine in the brains [9,21], thus it had been suggested that the inhibitory effect of galanin on cholinergic neurons worsened the degeneration of cognitive function in patients with AD. The detrimental role of galanin in AD was supported by the results that exogenous galanin impaired performance of a variety of cognitive tasks in rats [8]. However, galanin was shown to have biphasic effects when injected into rat hippocampus, with low dose of galanin (1 nmol/rat) improved the learning and memory ability of rats in water maze test, while it had opposite effect at high dose (3 nmol/rat) [24]. These results suggest that a homeostatic mechanism at play for the physiological or pathological role of galanin, this is supported by our present study showing that galanin regulated the M1 expression at a dose dependent manner in the primary cultured prefrontal cortical neurons. A second hypothesis is that galanin hyperinnervated the cholinergic neurons to promote neuronal function and survival as more recent studies demonstrated that galanin protected against amyloid beta induced toxicity in primary cultured basal forebrain neurons and hippocampal neurons [3,10]. Besides, galanin was found to attenuate kainate-induced excitotoxiciy in the cultured hippocampal slices [11]. Further study revealed that the protective effect of galanin excitotoxicity was mediated by galanin receptor 2 [12]. More interestingly, two independent groups demonstrated that administration of galanin into rat brains improved the spatial memory in the models of AD [3,19]. In our study, we demonstrated that galanin increased the expression of M1 in the primary cultured prefrontal cortical neurons. Our results are consistent with previous findings that galanin fiber hyperinnervation preserved neuroprotective gene expression and increased choline acetyltransferase expression in the cholinergic basal forebrain neurons of AD [5,6]. It has also been suggested that galanin hyperinnervation of cholinergic neurons regulates the tone of these perikarya allow-
ing them to continue to function in the late stage of AD [4,23]. Thus experiments using old neurons or amyloid precursor protein (APP) overexpressed neurons to study the regulatory effect of galanin on M1 expression would further elucidate the role of galanin in the progress of AD. Taken together, our data support the hypothesis that galanin acts as a trophic factor in response to the nerve injury during the onset of AD, further investigations into the therapeutic potential of galanin are justified.
Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (30870802, 81171043) and the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500706).
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Galanin up-regulates the expression of M1 muscarinic acetylcholine receptor via the ERK signaling pathway in primary cultured prefrontal cortical neurons Yong Cheng, Long-Chuan Yu ∗ Laboratory of Neurobiology and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China
h i g h l i g h t s • Galanin up-regulates the expression of M1 receptor in the neurons. • The ERK signaling pathway mediates the galanin induced up-regulation of M1 receptor. • Our study supports the hypothesis that galanin is neurotrophic in the progress of AD.
a r t i c l e
i n f o
Article history: Received 21 November 2014 Received in revised form 3 February 2015 Accepted 6 February 2015 Available online 9 February 2015 Key words: Galanin M1 muscarinic acetylcholine receptor Alzheimer’s disease ERK
a b s t r a c t The expression of galanin and galanin receptors are up-regulated in the brains from patients with Alzheimer’s disease (AD). However, the role of galanin in the progress of AD is still controversial. Here we demonstrated that galanin increased the protein expression of M1 muscarinic acetylcholine receptor (M1) in the primary cultured prefrontal cortical neurons by ELISA and Western Blot. Moreover, we showed that the mRNA expression of M1 was also up-regulated by galanin treatment. We further explored the mechanism of the galanin induced up-regulation of M1. We found that galanin activated the ERK signaling pathway in the primary cultured prefrontal cortical neurons. In addition, our results showed that the up-regulation of M1 mRNA was blocked by an ERK inhibitor, U0126. Taken together, our results demonstrated that the ERK signaling pathway mediated the galanin induced up-regulation of M1 in the primary cultured prefrontal cortical neurons, supporting the hypothesis that galanin plays a beneficial role in the development of AD. © 2015 Elsevier Ireland Ltd. All rights reserved.
Galanin is a 29 amino acids neuropeptide with wide expression in the central and peripheral nervous systems [18]. Galanin has been reported to be involved in many biological functions such as nociception, feeding, waking and sleeping regulation, learning and memory [16,18]. Moreover, galanin is linked to a number of neurological disorders, including epilepsy, depression and Alzheimer’s disease (AD) [18]. Galanin had been found to be up-regulated in the post-mortem brains of patients with AD more than twenty years ago [1]. However, the role of galanin during the onset of AD is still poorly understood. AD is a progressive neurodegenerative disease with amyloid beta and tau protein as two major hallmarks [14]. Another major hallmark for AD is the hypofunction of cholingergic
∗ Corresponding author. Tel.: +86 10 62753667; fax: +86 10 62751526. E-mail address: [email protected] (L.-C. Yu). http://dx.doi.org/10.1016/j.neulet.2015.02.011 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
system in the brain. It has been reported that there are reduction of acetylcholine synthesis and acetylcholine receptor level as well as significant loss of cholinergic neurons in the brains from patients with AD [15]. The neurotransmitter acetylcholine activates either ionotropic nicotinic acetycholine receptors or metabotrophic muscarinic acetylcholine receptors (mAChRs) to have biological functions [15]. Five mAChR (M1–M5) subtypes have been identified to date and they are involved in various physiological functions in the nervous system. Of the five mAChRs, M1 plays a crucial role in neurological diseases including AD [17]. It has been shown that activation of M1 attenuated amyloid beta induced neurotoxicity through the WNT signaling pathway [13]. Moreover, deficiency in M1 was found to further exacerbate AD-like pathology in a mouse model of AD [22]. Interestingly, a study found that a selective M1 agonist reduced amyloid beta and tau pathology in a transgenic mice model of AD [2]. Thus M1 has been postulated as an important therapeutic target for treating AD.
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Fig. 2. Galanin increased the expression of M1 mRNA in the neurons. Top panel: Representative RT-PCR analysis of M1 mRNA expression after galanin treatment at different time points in the neurons. GAPDH served as internal control. Bottom panel: Bar graphs showing the quantification of the M1 mRNA signals normalized to GAPDH. Note galanin increased the M1 mRNA expression at a time depend manner. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control.
Fig. 1. Galanin up-regulated the M1 protein expression in the primary cultured prefrontal cortical neurons. (A) Galanin increased the surface M1 expression in the primary cultured prefrontal cortical neurons as tested by ELISA assay. Note galanin had the maximum effect on the expression of surface M1 at the concentration of 1 nM. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control. (B) Top panel: Representative Western Blot analysis of total M1 protein expression after galanin treatment at different time points in the neurons. Actin served as internal control. Bottom panel: Bar graphs showing the quantification of the total M1 protein signals normalized to actin. Note galanin increased the total M1 protein expression at a time dependent manner. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control. Four duplicates were run for the Western Blot.
Although the regulatory role of galanin on the release of acetylcholine had been reported by many groups [9,21,25], there is no study showing whether galanin regulates the acetycholine receptors in the brains. Here we demonstrated that galanin up-regulated the expression of M1 in the primary cultured prefrontal cortical neurons, both in protein and mRNA levels. Furthermore, we found that the ERK signaling pathway mediated the galanin induced upregulation of M1. The primary cultured prefrontal cortical neurons of rats were prepared as described previously [27]. Briefly, the dissociated neurons from postnatal day zero to one pups (mixed gender) were plated at 105 cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 g/l HEPES, 2 g/l NaHCO3 , 100 U/mlstreptomycin and penicillin. To inhibit glial cell growth, cytosine arabinoside (10 M; Sigma, St.
Louis, Missouri) was supplemented after plating for 3 days. The neurons were used for experimentation after 7–8 days culture in vitro (DIV7-8). Galanin (Tocris, Bristol, UK) and 1,4-diamino-2,3dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126, Sigma, St. Louis, Missouri) were added freshly into the culture medium during treatments. U0126 is a highly selective inhibitor for extracellularsignal-regulated kinase 1/2 (ERK1/2). RT-PCRs were performed as described previously [20]. Briefly, primary cultured prefrontal cortical neurons were harvested and total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, California). Total RNA (2 ug) was reverse-transcribed using M-MLV Reverse Transcriptase (Invitrogen) and oligo-d(T) 15 random primers (Takara, Shiga, Japan). PCRs were done using Pre-mix Taq (Takara, Shiga, Japan). Sequences of the primers for the experiments were: rat M1: sense 5 -CAGTTCCTCTCCCAACCCAT3 and antisense 5 -TGGGCATCTTGATCACCACT-3 ; rat GAPDH: 5 -CGTATCGGACGCCTGGTT-3 and antisense 5 sense CCCTTCCACGATGCCAAAA-3 . Amplification was done at 94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min with a final extension cycle for 10 min at 72 ◦ C in an Eppendorf Master (Eppendorf, Hamburg, Germany). PCR products were electrophoresed on agarose gels, stained with ethidium bromide (Sigma, St. Louis, Missouri) and visualized under UV light. The results were assessed with a BioRad Chemi Doc XRS imaging system (BioRad Hercules, CA). Cell ELISA was used to detect M1 level on cell surface under non-permeabilized condition. The primary prefrontal cortical neurons of rats were treated and then gently washed with 0.01 M PBS before being fixed with 4% paraformaldehyde in PBS. Cells were then washed with PBS and blocked using PBS containing 10% donkey serum. After blocking, the cells were incubated with M1 primary antibody (1:200, Santa Cruz, CA, USA) diluted in 3% serum-PBS at 4 ◦ C overnight. Then cells were incubated with biotin-conjugated donkey anti-rabbit second antibody (1:1000) Jackson ImmunoResearch Laboratories, Inc., PA, USA) after washing with PBS, following by 0.3% H2 O2 incubation for 30 min to eliminate endogenous
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Fig. 3. The ERK1/2 signaling pathway mediated the galanin induced up-regulation of M1 in the neurons. (A) Top panel: Representative Western Blot analysis of p-ERK levels after 1 h or 3 h of galanin treatment in the primary cultured prefrontal cortical neurons. t-ERK served as internal control. Bottom panel: Bar graphs showing the quantification of p-ERK signals normalized to t-ERK 1/2. Note galanin activated the ERK signaling pathway in the neurons. Three duplicates were run for the Western Blot. (B) Top panel: Representative RT-PCR analysis of M1 mRNA expression after various treatments in the neurons. GAPDH served as internal control. Bottom panel: Bar graphs showing the quantification of M1 mRNA signals normalized to GAPDH. Note Galanin induced up-regulation of M1 mRNA expression was blocked by U0126 in the neurons, suggesting the ERK signaling pathway mediated the up-regulation of M1 induced by galanin. Values are mean ± SEM, one-way ANOVA followed Tukey post-hoc test, *p < 0.05, compared with control group; #p < 0.05 compared with galanin group.
peroxide enzyme. After washing with PBS, avidin–biotin complex (elite kit, Vector Laboratories, Burlingame, CA, USA) was added to the cells and incubated for 1 h. After washing, horseradish peroxidase (HRP) substrate 3,3 ,5,5 -tetramethylbenzidene (TMD) (0.1 mg/ml; Sigma, St. Louis, Missouri) was added to produce a color reaction that was stopped by 2N H2 SO4 . The optical density (absorbance) was read on a microplate reader (Model 550, Bio-Rad Hercules, CA) at 450 nm. Western Blot was performed as described previously [26,28]. Briefly, protein lysates of the primary cultured prefrontal cortical neurons were extracted after treatments. The lysates were iced for 30 min, centrifuged at 14,800×g for 15 min, and protein in the supernatant was harvested. The protein concentrations were determined using a spectrophotometric method. Denatured protein samples diluted with loading buffer were loaded equally into each lane, separated by 10% SDS-PAGE, and then blotted onto a polyvinylidene fluoride membrane. The membrane was then incubated for 1 h in blocking buffer (tris-buffered saline containing 5% defatted milk powder) at room temperature. Next, the membrane was incubated at 4 ◦ C with the primary antibodies, washed with tris-buffered saline and Tween-20 mixture and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by another wash. Immunoblots were developed in the presence of enhanced chemiluminescence reagents, and the images detected on X-ray film were quantified by densitometric scanning using a gel imaging analysis system. The purified polyclonal rabbit anti-t-ERK antibody (1:10000, Santa Cruz, CA, USA), polyclonal rabbit anti-M1 antibody (1:500, Santa Cruz, CA, USA), monoclonal mouse anti-p-ERK antibody (1:1000, Santa Cruz, CA, USA), polyclonal rabbit anti--actin antibody (1:3000, Santa Cruz, CA, USA),
HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, Inc., PA, USA), HRP-conjugated goat anti-mouse secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, Inc.), PVDF membrane (Millipore, MA, USA), X-ray film (Kodak, NY, USA) and Gel-Pro 4400 System (Media Cybernetics, Inc., MD, USA) were used in the experiments. Data were analyzed by one-way ANOVA followed by Tukey post-hoc multiple comparison test where noted. Significance was set at p < 0.05. To test whether galanin regulates the expression of M1, the primary cultured prefrontal cortical neurons were treated with vehicle, 100 pM, 1 nM,10 nM or 100 nM of galanin for 24 h, cell ELISA was then used to measure the expression level of M1 on cell membrane. As shown in Fig. 1A, galanin increased the expression of M1 on cell membrane at a dose dependent manner in the primary cultured prefrontal cortical neurons (ANOVA between control and treated groups, F(4,94) = 3.742, n = 20, p < 0.05), compared to the control group. Since the above results indicated that the effect of galanin on the expression of M1 peaks at 1 nM, we therefore used 1 nM of galanin in the subsequent experiments. We then used western blot to measure the total levels of M1 in primary cultured prefrontal cortical neurons. The neurons were treated with vehicle or 1 nM of galanin for 6 h, 12 h or 24 h and protein samples were collected for analysis. Results from western blot showed that the expression of total M1 was increased by galanin incubation (ANOVA between control and treated groups, F(3,15) = 5.819, n = 4, p < 0.05) in the neurons, compared to the control group, as shown in Fig. 1B. Our data have shown that galanin up-regulated the expression of M1 protein in the primary cultured prefrontal cortical
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neurons, we then tested mRNA expression of M1 after galanin treatment in the neurons. Results from RT-PCR demonstrated that the expression of M1 mRNA was increased after 6 h, 12 h, or 24 h of galanin treatment (ANOVA between control and treated groups, F(3,11) = 4.848, n = 4, p < 0.05) in the neurons, compared to the control group, as shown in Fig. 2. To elucidate the mechanism by which galanin mediated the synthesis of M1 in the neurons, we searched for the potential downstream signaling pathways that are known to be involved in the regulations of genes. We tested if galanin can activate the ERK signaling pathway, a signaling pathway that regulates gene expression and has been shown to be activated by galanin in hippocampus [12]. As showed in Fig. 3A, galanin increased the level of phosphorylated ERK after 1 h or 3 h treatment in the primary cultured prefrontal cortical neurons (ANOVA between control and treated groups, F(2,8) = 19.28, n = 3, p < 0.05), compared to the control group. However, the total ERK levels were not changed after galanin treatment, suggesting that galanin activated the ERK signaling pathway in the primary cultured prefrontal cortical neurons. To further investigate whether galanin induced up-regulation of M1 was mediated by the ERK signaling pathway in the neurons, we used the ERK inhibitor, U0126. Results from RT-PCR showed that pretreatment with 1 M U0126 for 30 min in the neurons completely blocked the increased expression of M1 mRNA induced by galanin (ANOVA between control and treated groups, F(2,8) = 9.67, n = 3, p < 0.05), as shown in Fig. 3B. The above results indicated that galanin induced up-regulation of M1 in the primary cultured prefrontal cortical neurons was mediated by the ERK signaling pathway. An important feature of AD is the increased expression of galanin and galanin receptors in the post-mortem brains of patients with AD [7]. Galanin was further found to be hyperinnervating the surviving cholinergic neurons in the brains from patients with AD [7]. Various studies showed that galanin had an inhibitory role in the release of acetycholine in the brains [9,21], thus it had been suggested that the inhibitory effect of galanin on cholinergic neurons worsened the degeneration of cognitive function in patients with AD. The detrimental role of galanin in AD was supported by the results that exogenous galanin impaired performance of a variety of cognitive tasks in rats [8]. However, galanin was shown to have biphasic effects when injected into rat hippocampus, with low dose of galanin (1 nmol/rat) improved the learning and memory ability of rats in water maze test, while it had opposite effect at high dose (3 nmol/rat) [24]. These results suggest that a homeostatic mechanism at play for the physiological or pathological role of galanin, this is supported by our present study showing that galanin regulated the M1 expression at a dose dependent manner in the primary cultured prefrontal cortical neurons. A second hypothesis is that galanin hyperinnervated the cholinergic neurons to promote neuronal function and survival as more recent studies demonstrated that galanin protected against amyloid beta induced toxicity in primary cultured basal forebrain neurons and hippocampal neurons [3,10]. Besides, galanin was found to attenuate kainate-induced excitotoxiciy in the cultured hippocampal slices [11]. Further study revealed that the protective effect of galanin excitotoxicity was mediated by galanin receptor 2 [12]. More interestingly, two independent groups demonstrated that administration of galanin into rat brains improved the spatial memory in the models of AD [3,19]. In our study, we demonstrated that galanin increased the expression of M1 in the primary cultured prefrontal cortical neurons. Our results are consistent with previous findings that galanin fiber hyperinnervation preserved neuroprotective gene expression and increased choline acetyltransferase expression in the cholinergic basal forebrain neurons of AD [5,6]. It has also been suggested that galanin hyperinnervation of cholinergic neurons regulates the tone of these perikarya allow-
ing them to continue to function in the late stage of AD [4,23]. Thus experiments using old neurons or amyloid precursor protein (APP) overexpressed neurons to study the regulatory effect of galanin on M1 expression would further elucidate the role of galanin in the progress of AD. Taken together, our data support the hypothesis that galanin acts as a trophic factor in response to the nerve injury during the onset of AD, further investigations into the therapeutic potential of galanin are justified.
Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (30870802, 81171043) and the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500706).
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