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Liraglutide Protects Neurite Outgrowth of Cortical Neurons Under Oxidative Stress though Activating the Wnt Pathway
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Liraglutide Protects Neurite Outgrowth of Cortical Neurons Under Oxidative Stress though Activating the Wnt Pathway TagedPD1X XWeiliang He, D2XMD, X * D3X XXiaochao Tian, MD, D4X X † D5X XMimi Lv, D6XMD, X * and D7X XHebo Wang, D8XMD X * TagedP
Background: Neurogenesis including neurite outgrowth is important for brain plasticity under physiological conditions and in brain repair after injury. Liraglutide has been found to have neuroprotective action in the risk of central nervous system disease. However, the effect and the potential mechanism of liraglutide-induced neurite outgrowth in primary cortical neurons under oxidative stress remain poorly documented. Methods: In the text, H2O2 was used to mimic ischemia injury in primary cortical neurons. The viability and apoptosis of cell was assessed by Cell Counting Kit-8 and Hoechst 33342. Immunofluorescence method was used to examine the effect of liraglutide on neurite outgrowth in cortical neuron under H2O2 condition. Then, the potential mechanisms involving the Wnt pathway were investigated. The expression of b-catenin, c-myc, and cyclin D1 was determined using quantitative real-time polymerase chain reaction and Western blot. Results: Liraglutide significantly increased the viability and alleviated the apoptosis rate of cortical neurons induced by H2O2. Next, liraglutide promoted neurite outgrowth, which could be partially inhibited by the Wnt pathway inhibitor Xav939. Besides, liraglutide induced an increase of b-catenin, c-myc, and cyclin D1 levels, which could also be blocked in the presence of Xav939. Conclusions: These results illustrate that liraglutide exerts neurotrophin-like activity in cortical neurons under oxidative stress condition, partly through activating the Wnt pathway. K TagedP ey Words: Cerebral ischemia—liraglutide—cortical neuron—neurite outgrowth— the Wnt pathway. © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction TagedPNeuroplasticity is used to describe a range of adaptive changes occurred in the structure and function of cells in the nervous system.1 Neurogenesis including neurite outgrowth is a critical step in neuronal development and regeneration, and essential for forming functional neural circuits, which is believed to be an important mechanism of brain plasticity under physiological conditions and in brain repair after injury.2-4 Thus, the identification of novel drugs inducing neurite outgrowth for the treatment From the *Department of Neurology, Hebei General Hospital, Shijiazhuang, Hebei, PR China; and †Department of Cardiology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, PR China. Received March 30, 2018; revision received May 20, 2018; accepted May 25, 2018. Address correspondence to Hebo Wang, MD, Department of Neurology, Hebei General Hospital, Shijiazhuang, Hebei 050000, PR China. E-mail: [email protected] 1052-3057/$ - see front matter © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2018.05.039
TagedP f diseases of the human central nervous system (CNS) is o imperative. TagedPLiraglutide, a glucagon-like peptide-1(GLP-1) synthetic form, has been approved by the US Food and Drug Administration as a therapy for treatment of type 2 diabetes.5 Recent studies have shown that liraglutide has been found to have neuroprotective action in ischemic stroke, Alzheimer's disease, and Parkinson's disease.6-9 Li et al also showed that liraglutide displayed promoting neuritogenic activity role in primary cortical neurons.10 In addition, GLP-1 and Exendin-4, a GLP-1 agonist, have been reported to play a role in neuronal survival, neurite outgrowth, and synaptic plasticity in vitro.11,12 Those phenomenon may contribute to the reduction in the risk of CNS disease in those taking GLP-1. Activation of RhoA and MEK-extracellular signal-regulated kinases (ERK) pathway induced by GLP-1 or its agonist has been shown to regulate neurite outgrowth.10,13 However, the mechanism of liraglutideinduced neurite outgrowth remains poorly documented, but they are vital to understand neuroplasticity. TagedPThe Wnt pathway plays an effect in many developmental processes such as neuronal maturation, migration,
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T euronal connectivity, and synaptic formation.14 ActivatagedPn ing Wnt pathway stimulates neural stem cell proliferation and self-renewal.15 Several studies in various species have established that Wnts and their receptors have an important role in regulating neurite outgrowth and guiding axons.16 The Wnt pathway has been shown to be involved in all different stages of adult neurogenesis in the subgranular zone.17 Wnt stimulation of neurite outgrowth concerned axonal remodeling in cultured mouse cerebellar granule cells.18 In addition, GLP-1 exerts proliferative and protective effects on pancreatic b-cells, which are involved in the Wnt pathway.19 However, whether the Wnt pathway involved liraglutide-mediated neurite outgrowth needs to be further investigated. TagedPTherefore, the aim of this present study is to assess the potential effects of liraglutide on neurite outgrowth of cortical neurons in H2O2-induced neuronal injury in vitro and the role of the Wnt pathway on liraglutide-induced neurite growth.
Materials and Methods Animals T 57BL/6 mice were purchased from Vital River LaboagedPC ratory Animal Technology Co. Ltd. (Beijing, China). The protocol was approved by the Institutional Animal Care and Use Committee and the Local Experimental Ethics Committee. And this study conforms to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health.
Primary Cortical Neuron Culture TagedPCortical neurons were obtained from the embryonic brains at embryonic days 15-18 (E15-18) of C57BL/6 mice. The cerebral cortex of the embryonic brains was dissected under an optical microscope (Olympus, Tokyo, Japan) and incubated in papain (2 mg/ml) (Sigma, St. Louis, MO) in Hibernate-E (Sigma) for 15 minutes at 37°C. Then the dispersed tissues were neutralized with Hibernate-E and dissociated into a single cell. Neurons were plated onto culture dishes coated with poly-L-lysine (Biocoat, BD Biosciences, San Jose, CA, USA), grown in neurobasal medium containing 2% B-27 supplement (Invitrogen, Waltham, MA, USA) and 0.5 mM glutamine (Life Technologies, Waltham, MA, USA), and incubated in a humidified 5% CO2 incubator at 37°C.
Pharmacological Treatment TagedPH2O2 is a precursor to hydroxyl radicals, which mimicked ischemia injury,20 so we used 100 mM H2O2 (Sigma) to conduct the following experiment. To evaluate the effect of liraglutide on neurite outgrowth of cortical neurons under oxidative stress, liraglutide (Novo Nordisk, Copenhagen, Denmark) was used to treat neurons. Neurons were pretreated with 100 mM H2O2 for 2 hours, subsequently with the treatment of liraglutide (1/10/100/
1TagedP 000 nM) for 24 hours. The control group was performed with no liraglutide or H2O2 treatment. TagedPTo further assess the effect of the Wnt pathway, the Wnt pathway inhibitor, Xav939 (0.5 mM) (Sigma) was added to neurons 30 minutes before liraglutide treatment.
Cell Viability Assay TagedPThe viability of cortical neurons was evaluated using Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). At 24 hours after indicated treatment, 10 ml CCK-8 was added to each well, and the plates were incubated for additional 2 hours. Optical Density (OD) at a wavelength of 450 nm was measured by a microtiter plate reader (Tecan, Switzerland). These values were represented as OD values as percent of control. Each group was done with 6 replicates (n = 6), and the experiment was conducted 3 times. Additional, the apoptosis of neurons was detected by Hoechst 33342 at a final concentration of 5 mg/ml. Cells were observed immediately with a fluorescence microscope (Olympus, Tokyo, Japan).
Determination of Neurite Outgrowth TagedPAfter 24 hours culture of indicated treatment, primary cortical neurons were fixed with 4% paraformaldehyde for 20 minutes, and then permeabilized using 0.3% Triton-X-phosphate-buffered saline for 15 minutes at room temperature. After blocking nonspecific binding with phosphate-buffered saline containing 1% bovine serum albumin (immunoglobulin G-free; Sigma) for 1 hour. Neurons were then incubated with mouse monoclonal antib-III-tubulin antibody (TuJ-1, 1:500, Sigma) overnight at 4°C, followed by donkey antimouse immunoglobulin G, tetramethylrhodamine, conjugated (1:200, CWBIO, Beijing, China) for 1 hour at 37°C. The images were captured with a fluorescence microscope (Olympus, Tokyo). The length of the longest neurite was measured using Image J software. The experiment was conducted 3 times.
Quantitative Real-Time Polymerase Chain Reaction TagedPAfter 24 hours culture of indicated treatment, Cells were harvested. The total RNA of neurons was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). Firststrand cDNA was reverse transcribed using the Firststrand cDNA synthesis kit (Fermentas International Inc. Waltham, MA, USA). The cDNA was amplified by realtime polymerase chain reaction (PCR) using a real-time PCR system (Applied Biosystems) in the presence of SYBR Green mix (Fermentas, Glen Burnie, MD). The realtime PCR was carried out at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, and final extension at 72° C for 7 minutes for 40 cycles. The determinations of relative b-catenin, c-myc, and cyclin D1 mRNA expression were made with 2¡delta delta CT method. Primers for realtime PCR were listed as follow according to previously reported21,22:
ARTICLE IN PRESS LIRAGLUTIDE PROTECTS NEURITE OUTGROWTH OF CORTICAL NEURONS
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b-catenin: sense: 50 -GATTTGATGGAGTTGGACATGG-30 , antisense: 50 -TGTTCTTGAGTGAAGGACTGAG-30 ; b-catenin: sense: 50 -TAACTCGAGGAGGAGCTGGA-30 , antisense: 50 -GCCAAGGTTGTGAGGTTAGG-30 ; cyclin D1: sense: 50 -TGGAGCCCCTGAAGAAGAG-30 , antisense: 50 -AAGTGCGTTGTGCGGTAGC-30 ; b-actin: sense: 50 -GGCATCGTGATGGACTCCG-30 , antisense: 50 -GCTGGAAGGTGGACAGCGA-30 . Western Blot
TagedPProteins of cultured neurons were extracted according to the manufacturer's instructions (Applygen Technologies Inc., Beijing) and quantified with the Bicinchoninic Acid Protein Assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA). Equal proteins were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with the following antibodies: anti-b-catenin (1:200; Cell Signaling Technology, Danvers, MA, USA), anti-c-myc (1:200; Cell Signaling Technology, Danvers, MA, USA), and anti-cyclin D1 (1:200; Cell Signaling Technology, Danvers, MA, USA). After washing with Tris-buffered saline and 0.1% Tween,
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TagedPthe membranes were incubated with Tris-buffered saline and 0.1% Tween -containing fluorescent-labeled secondary antibodies (IRDye; 800-conjugated goat anti-mouse 1:10,000 dilution; Rockland, Gilbertsville, PA) for 1 hour. The bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). A mouse anti glyceraldehyde-3phosphate dehydrogenase (1:5000; Sigma) was used as an internal control.
Statistical Analysis TagedPData were presented as mean § SEM. One-way analysis of variance followed by Student Newman Keuls (S-N-K) posthoc analysis was performed with SPSS V.17.0. P < 0.05 was considered to be statistically significant.
Results Liraglutide Increases the Viability of Cortical Neurons Damaged by Oxidative Stress TagedPFirst, we established whether liraglutide protected cortical neurons under oxidative stress using CCK-8 assays. Results showed that H2O2 decreased the cell viability by approximately 70.82% compared with the control group; the decrease in cell viability was rescued by liraglutide treatment. Moreover, we observed that liraglutide could increase the viability in a dose-dependent manner (Fig 1,
Figure 1. Liraglutide restores cell viability in primary cultured cortical neurons damaged by oxidative stress. Neurons were pretreated with 100 mM H2O2 for 2 hours, subsequently with the treatment of different concentrations of liraglutide for 24 hours. (A) The cell viability was determined by CCK-8 assay. Each group was done with 6 replicates (n = 6), and the experiment was replicated 3 times. Depicted were mean § S.E.M., *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group represents statistic difference. (B) The cell apoptosis was determined by Hoechst 33342 staining. Bar = 50 mm. Depicted were mean § S.E.M., *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group represents statistic difference. Abbreviation: CCK-8, Cell Counting Kit-8
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TagedPA). The viability of cortical neurons was in 31.16 § 4.09%, 40.09 § 3.82%, 63.61 § 5.11%, 53.22 § 5.72% in 1/10/ 100/1000 nM liraglutide group under oxidative stress, respectively. Results revealed that liraglutide could significantly protect neurons against oxidative stress. Considering the effectiveness, 100 nM liraglutide might be suitable to conduct the remaining experiments. TagedPCell apoptosis was also assessed by Hoechst 33342. As shown in Figure 1B, the percentage of the apoptosis cells in control group, H2O2 group, and H2O2 + liraglutide group was 12.20 § 2.05%, 73.05 § 6.63%, and 43.18 § 4.35%, respectively, which suggested that liraglutide could alleviate the H2O2-induced apoptosis of neurons.
Liraglutide Protects Neuritogenesis Ability of Cortical Neurons Insulted by Oxidative Stress TagedPNext, We explored whether liraglutide promoted neurite outgrowth OD cortical neurons under oxidative stress evaluated by measuring the longest neurite. As shown in Figure 2, neurons exposed to H2O2 showed a significant decrease in neurite outgrowth (22.88 § 2.82 mm), compared with the control group (35.54 § 5.21 mm). However, liraglutide significantly recovered the decrease in neurite outgrowth, (30.30 § 2.97 mm). These results suggested that liraglutide could effectively protect neuritogenesis of neurons subjected to oxidative injury.
A Wnt Pathway Inhibitor Xav939 Inhibits LiraglutideInduced Neurite Elongation Under Oxidative Stress TagedPWe further investigated the underlying molecule mechanism in liraglutide-induced neurite elongation under
TagedP xidative stress. Previous studies have shown the Wnt o pathway has been involved in neurogenesis in the subgranular zone.17 In addition, GLP-1 exerts proliferative and protective effects involving the Wnt signaling pathway.19 Therefore, we hypothesize that the Wnt pathway was involved in liraglutide-induced neurite elongation. To test this hypothesis, the Wnt pathway inhibitor Xav939 was used to block the Wnt pathway. As shown in Figure 2, Xav939 significantly prevented liraglutide-mediated neurite outgrowth of cortical neurons under H2O2. These data indicate that liraglutide restored neurite outgrowth of cortical neurons under oxidative stress partially blocked by Xav939.
The Wnt Pathway is Required for Liraglutide-Induced Neurite Outgrowth Under Oxidative Stress TagedPSince Xav939 could counteract the promoting effect of liraglutide on neurite outgrowth of cortical neurons under oxidative stress, and b-catenin, c-myc, and cyclin D1 were downstream target molecules of the Wnt pathway, we then sought to determine whether b-catenin, c-myc, and cyclin D1 were involved in liraglutide-induced neurite outgrowth. The genes and proteins levels of b-catenin, cmyc, and cyclin D1 were measured using quantitative real-time polymerase chain reaction and Western blot assays. Compared with the control group, the genes and proteins expressions of b-catenin, c-myc, and cyclin D1 were significantly decreased in H2O2 group. However, liraglutide significant increased the b-catenin, c-myc, and cyclin D1 levels. In addition, we also observed Xav939 could prevent the increased b-catenin, c-myc, and cyclin D1 levels that liraglutide induced (Figs 3 and 4). These indicate that the Wnt pathway play an effect in
Figure 2. Xav939 inhibits liraglutide-induced neurite elongation of cortical neurons against oxidative stress. Bar = 20 mm. (A) Representative images of cultured cortical neurons stained with anti-b-tubulin antibody in different treatment. (a) Cont: neurons cultured in medium without H2O2. (b) Neurons cultured with H2O2. (c) Neurons cultured with H2O2 and 100 nM liraglutide. (d) Neurons cultured with H2O2 and 100 nM liraglutide and 0.5 mM Xav939. (B) Statistic results in different groups were shown as above chart. Values represent the mean § S.E.M. (n = 60 neurons). *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference.
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Figure 3. qRT-PCR analysis of the expressions of b-catenin, c-myc, and cyclin D1 mRNAs in different experimental conditions. Liraglutide promotes neurite outgrowth insulted by H2O2 by mediating the Wnt pathway. (A) b-catenin; (B) c-myc; and (C) cyclin D1. Bar graphs with error bars represent mean § SEM. *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference. Abbreviation: qRT-PCR, quantitative real-time polymerase chain reaction.
TagedPliraglutide-mediated neurite outgrowth of cortical neurons under oxidative stress.
Discussion TagedPIn this study, we examined the effect of liraglutide on neurite outgrowth under oxidative stress and the underlying mechanism. To our knowledge, we demonstrated for the first time that liraglutide promoted neuroprotective and neurotropic ability of cortical neurons insulted by oxidative stress partly involved in the Wnt pathway. TagedPBecause diabetes has been identified as a risk factor for disorders of CNS, drug-treated diabetes may be useful to
TagedP ave a neuroprotective effect.23 GLP-1, an intestinalh secreted incretin, can decrease blood sugar by increasing cellular glucose uptake. Recent studies suggest that GLP1 acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, protects synaptic plasticity, and memory formation and reduces plaque formation and the inflammation response.11,12 The emerging data show that GLP-1 and its agonist extendin-4 have neurotrophic function to induce neuronal differentiation of PC12 cells and prevent neurons damage challenged by oxidative stress.24 Moreover, Exendin-4 increased neurite outgrowth of adult sensory neurons in vitro.12 However, liraglutide is a newer GLP-1 analog and also has been
Figure 4. Western blot analysis of the expressions of b-catenin and cyclin D1 proteins in different experimental conditions. (A) Western blot for b-catenin protein levels in each group, (B) bar graph shows the mean § S.E.M. of b-catenin/GAPDH ratio. (C) Western blot for c-myc protein levels in each group, (D) bar graph shows the mean § S.E.M. of c-myc/GAPDH ratio. (E) Western blot for cyclin D1 protein levels in each group, (F) bar graph shows the mean § S.E.M. of cyclin D1/GAPDH ratio. *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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agedPrT eleased onto the market several years ago.5 It has a longer biological half-life, but also can cross the blood-brain barrier,25,26 which are of vital importance for treat disorders of the CNS. Liraglutide has been found to have neuroprotective action in various experimental models. Liraglutide significantly decreased the infarct volume and improved neurologic deficits in a rat model of middle cerebral artery occlusion.6 Liraglutide exerts protective effects on memory formation, synaptic plasticity, synapse survival, and a reduction of amyloid synthesis and plaque load in the brain.27 Liraglutide significantly increased memory retention and total hippocampal CA1 pyramidal neuron numbers in SAMP8 mice.9 In addition, Li et al reported that liraglutide promoted neurite outgrowth of primary cortical neurons in vitro.10 Here, liraglutide treatment rescued the viability and decreased apoptosis of neurons insulted by H2O2 in vitro model. The neurite outgrowth assay further illustrated liraglutide could exert a neuroprotective effect on neurite outgrowth of cortical neurons against oxidative stress. These findings suggested that liraglutide could block cellular injury and promote neurite outgrowth ability of cortical neurons under oxidative stress. TagedPThe Wnt pathway is important in regulation of many cellular events, including prevention of cellular apoptosis as well as promotion of cell regeneration.28 In brain, the Wnt pathway not only serves to foster neuronal survival,29 but also be associated with axonal remodeling, dendritic morphogenesis, and neuronal connectivity.30-32 The Wnt pathway has been shown to regulate neurite outgrowth by increasing neurite spreading and branching as well as expression of synapsin I, a protein that participates in synapse formation and neurotransmitter release in primary neuronal cell culture.33 Targeted disruption of the Fzd3 gene, one of the Wnt pathway genes, in mouse produced severe defects in several major axon tracts of the forebrain.34 Over-expression of b-catenin has inhibited neurite outgrowth in embryonic mouse retinal explant cultures and PC12 cells.35,36 In addition, a conserved Wnt pathway which is crucial for anterior-posterior outgrowth of neurites from RME head motor neurons in Caenorhabditis elegans important role of a Wnt-Frz/Ror-Dsh pathway in regulating neurite anterior-posterior outgrowth.37 Dishevelled-2/3 mediates Wnt-3a-dependent neurite outgrowth in Ewing sarcoma family tumor cells.38 It is reported that GLP-1 is a direct activator of the Wnt pathway.39 Therefore, the Wnt pathway implicated in the regulation of liraglutide-induced neurite outgrowth could be of great potential to evaluate the underlying mechanism, in our study, treatment with a special Wnt pathway inhibitor Xav939 partially inhibited the enhancing effect played by liraglutide in neurite outgrowth. Consistently, the gene and protein levels of b-catenin, c-myc, and cyclin D1 were significantly increased by the liraglutide treatment, however, partially reduced by Xav939. These data indicate that the Wnt pathway may at least partially contribute to
lTagedP iraglutide-induced neuritogenesis of cortical neurons under H2O2, which may provide a new insight to promote brain plasticity. TagedPIn conclusion, we have shown that liraglutide restored the viability, inhibited apoptosis, and protected neurite outgrowth of cortical neurons under oxidative stress. One of the related mechanisms may be the activating of the Wnt pathway. But more experiments should be carried out to further clarify the exact mechanisms. In addition, it needs to be further determined that whether the conclusion in this study can also apply to animals or clinical trials.
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TagedPin the C57B/16 mouse brain. Brain Res 2012;1473:204213. Briyal S, Shah S, Gulati A. Neuroprotective and antiapoptotic effects of liraglutide in the rat brain following focal cerebral ischemia. Neuroscience 2014;281:269-281. Lee JW, Lee YK, Yuk DY, et al. Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta amyloid generation. J Neuroinflammation 2008;5:37. Chong ZZ, Maiese K. Targeting WNT, protein kinase B, and mitochondrial membrane integrity to foster cellular survival in the nervous system. Histol Histopathol 2004; 19:495-504. Hall AC, Lucas FR, Salinas PC. Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 2000;100:525-535. Rosso SB, Sussman D, Wynshaw-Boris A, et al. Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development. Nat Neurosci 2005;8:34-42. Ciani L, Salinas PC. WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nat Rev Neurosci 2005;6:351-362. Ille F, Sommer L. Wnt signaling: multiple functions in neural development. Cell Mol Life Sci 2005;62:1100-1108. Wang Y, Thekdi N, Smallwood PM, et al. Frizzled-3 is required for the development of major fiber tracts in the rostral CNS. J Neurosci 2002;22:8563-8573. Ouchi Y, Tabata Y, Arai K, et al. Negative regulation of retinal-neurite extension by beta-catenin signaling pathway. J Cell Sci 2005;118:4473-4483. Votin V, Nelson WJ, Barth AI. Neurite outgrowth involves adenomatous polyposis coli protein and betacatenin. J Cell Sci 2005;118:5699-5708. Song S, Zhang B, Sun H, et al. A Wnt-Frz/Ror-Dsh pathway regulates neurite outgrowth in Caenorhabditis elegans. PLoS Genet 2010: 6. pii: e1001056. Greer YE, Rubin JS. Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth. J Cell Biol 2011;192:993-1004. Zhao C, Liang J, Yang Y, et al. The impact of Glucagon-Like Peptide-1 on bone metabolism and its possible mechanisms. Front Endocrinol (Lausanne) 2017;8:98.
Liraglutide Protects Neurite Outgrowth of Cortical Neurons Under Oxidative Stress though Activating the Wnt Pathway TagedPD1X XWeiliang He, D2XMD, X * D3X XXiaochao Tian, MD, D4X X † D5X XMimi Lv, D6XMD, X * and D7X XHebo Wang, D8XMD X * TagedP
Background: Neurogenesis including neurite outgrowth is important for brain plasticity under physiological conditions and in brain repair after injury. Liraglutide has been found to have neuroprotective action in the risk of central nervous system disease. However, the effect and the potential mechanism of liraglutide-induced neurite outgrowth in primary cortical neurons under oxidative stress remain poorly documented. Methods: In the text, H2O2 was used to mimic ischemia injury in primary cortical neurons. The viability and apoptosis of cell was assessed by Cell Counting Kit-8 and Hoechst 33342. Immunofluorescence method was used to examine the effect of liraglutide on neurite outgrowth in cortical neuron under H2O2 condition. Then, the potential mechanisms involving the Wnt pathway were investigated. The expression of b-catenin, c-myc, and cyclin D1 was determined using quantitative real-time polymerase chain reaction and Western blot. Results: Liraglutide significantly increased the viability and alleviated the apoptosis rate of cortical neurons induced by H2O2. Next, liraglutide promoted neurite outgrowth, which could be partially inhibited by the Wnt pathway inhibitor Xav939. Besides, liraglutide induced an increase of b-catenin, c-myc, and cyclin D1 levels, which could also be blocked in the presence of Xav939. Conclusions: These results illustrate that liraglutide exerts neurotrophin-like activity in cortical neurons under oxidative stress condition, partly through activating the Wnt pathway. K TagedP ey Words: Cerebral ischemia—liraglutide—cortical neuron—neurite outgrowth— the Wnt pathway. © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction TagedPNeuroplasticity is used to describe a range of adaptive changes occurred in the structure and function of cells in the nervous system.1 Neurogenesis including neurite outgrowth is a critical step in neuronal development and regeneration, and essential for forming functional neural circuits, which is believed to be an important mechanism of brain plasticity under physiological conditions and in brain repair after injury.2-4 Thus, the identification of novel drugs inducing neurite outgrowth for the treatment From the *Department of Neurology, Hebei General Hospital, Shijiazhuang, Hebei, PR China; and †Department of Cardiology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, PR China. Received March 30, 2018; revision received May 20, 2018; accepted May 25, 2018. Address correspondence to Hebo Wang, MD, Department of Neurology, Hebei General Hospital, Shijiazhuang, Hebei 050000, PR China. E-mail: [email protected] 1052-3057/$ - see front matter © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2018.05.039
TagedP f diseases of the human central nervous system (CNS) is o imperative. TagedPLiraglutide, a glucagon-like peptide-1(GLP-1) synthetic form, has been approved by the US Food and Drug Administration as a therapy for treatment of type 2 diabetes.5 Recent studies have shown that liraglutide has been found to have neuroprotective action in ischemic stroke, Alzheimer's disease, and Parkinson's disease.6-9 Li et al also showed that liraglutide displayed promoting neuritogenic activity role in primary cortical neurons.10 In addition, GLP-1 and Exendin-4, a GLP-1 agonist, have been reported to play a role in neuronal survival, neurite outgrowth, and synaptic plasticity in vitro.11,12 Those phenomenon may contribute to the reduction in the risk of CNS disease in those taking GLP-1. Activation of RhoA and MEK-extracellular signal-regulated kinases (ERK) pathway induced by GLP-1 or its agonist has been shown to regulate neurite outgrowth.10,13 However, the mechanism of liraglutideinduced neurite outgrowth remains poorly documented, but they are vital to understand neuroplasticity. TagedPThe Wnt pathway plays an effect in many developmental processes such as neuronal maturation, migration,
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T euronal connectivity, and synaptic formation.14 ActivatagedPn ing Wnt pathway stimulates neural stem cell proliferation and self-renewal.15 Several studies in various species have established that Wnts and their receptors have an important role in regulating neurite outgrowth and guiding axons.16 The Wnt pathway has been shown to be involved in all different stages of adult neurogenesis in the subgranular zone.17 Wnt stimulation of neurite outgrowth concerned axonal remodeling in cultured mouse cerebellar granule cells.18 In addition, GLP-1 exerts proliferative and protective effects on pancreatic b-cells, which are involved in the Wnt pathway.19 However, whether the Wnt pathway involved liraglutide-mediated neurite outgrowth needs to be further investigated. TagedPTherefore, the aim of this present study is to assess the potential effects of liraglutide on neurite outgrowth of cortical neurons in H2O2-induced neuronal injury in vitro and the role of the Wnt pathway on liraglutide-induced neurite growth.
Materials and Methods Animals T 57BL/6 mice were purchased from Vital River LaboagedPC ratory Animal Technology Co. Ltd. (Beijing, China). The protocol was approved by the Institutional Animal Care and Use Committee and the Local Experimental Ethics Committee. And this study conforms to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health.
Primary Cortical Neuron Culture TagedPCortical neurons were obtained from the embryonic brains at embryonic days 15-18 (E15-18) of C57BL/6 mice. The cerebral cortex of the embryonic brains was dissected under an optical microscope (Olympus, Tokyo, Japan) and incubated in papain (2 mg/ml) (Sigma, St. Louis, MO) in Hibernate-E (Sigma) for 15 minutes at 37°C. Then the dispersed tissues were neutralized with Hibernate-E and dissociated into a single cell. Neurons were plated onto culture dishes coated with poly-L-lysine (Biocoat, BD Biosciences, San Jose, CA, USA), grown in neurobasal medium containing 2% B-27 supplement (Invitrogen, Waltham, MA, USA) and 0.5 mM glutamine (Life Technologies, Waltham, MA, USA), and incubated in a humidified 5% CO2 incubator at 37°C.
Pharmacological Treatment TagedPH2O2 is a precursor to hydroxyl radicals, which mimicked ischemia injury,20 so we used 100 mM H2O2 (Sigma) to conduct the following experiment. To evaluate the effect of liraglutide on neurite outgrowth of cortical neurons under oxidative stress, liraglutide (Novo Nordisk, Copenhagen, Denmark) was used to treat neurons. Neurons were pretreated with 100 mM H2O2 for 2 hours, subsequently with the treatment of liraglutide (1/10/100/
1TagedP 000 nM) for 24 hours. The control group was performed with no liraglutide or H2O2 treatment. TagedPTo further assess the effect of the Wnt pathway, the Wnt pathway inhibitor, Xav939 (0.5 mM) (Sigma) was added to neurons 30 minutes before liraglutide treatment.
Cell Viability Assay TagedPThe viability of cortical neurons was evaluated using Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). At 24 hours after indicated treatment, 10 ml CCK-8 was added to each well, and the plates were incubated for additional 2 hours. Optical Density (OD) at a wavelength of 450 nm was measured by a microtiter plate reader (Tecan, Switzerland). These values were represented as OD values as percent of control. Each group was done with 6 replicates (n = 6), and the experiment was conducted 3 times. Additional, the apoptosis of neurons was detected by Hoechst 33342 at a final concentration of 5 mg/ml. Cells were observed immediately with a fluorescence microscope (Olympus, Tokyo, Japan).
Determination of Neurite Outgrowth TagedPAfter 24 hours culture of indicated treatment, primary cortical neurons were fixed with 4% paraformaldehyde for 20 minutes, and then permeabilized using 0.3% Triton-X-phosphate-buffered saline for 15 minutes at room temperature. After blocking nonspecific binding with phosphate-buffered saline containing 1% bovine serum albumin (immunoglobulin G-free; Sigma) for 1 hour. Neurons were then incubated with mouse monoclonal antib-III-tubulin antibody (TuJ-1, 1:500, Sigma) overnight at 4°C, followed by donkey antimouse immunoglobulin G, tetramethylrhodamine, conjugated (1:200, CWBIO, Beijing, China) for 1 hour at 37°C. The images were captured with a fluorescence microscope (Olympus, Tokyo). The length of the longest neurite was measured using Image J software. The experiment was conducted 3 times.
Quantitative Real-Time Polymerase Chain Reaction TagedPAfter 24 hours culture of indicated treatment, Cells were harvested. The total RNA of neurons was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). Firststrand cDNA was reverse transcribed using the Firststrand cDNA synthesis kit (Fermentas International Inc. Waltham, MA, USA). The cDNA was amplified by realtime polymerase chain reaction (PCR) using a real-time PCR system (Applied Biosystems) in the presence of SYBR Green mix (Fermentas, Glen Burnie, MD). The realtime PCR was carried out at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, and final extension at 72° C for 7 minutes for 40 cycles. The determinations of relative b-catenin, c-myc, and cyclin D1 mRNA expression were made with 2¡delta delta CT method. Primers for realtime PCR were listed as follow according to previously reported21,22:
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b-catenin: sense: 50 -GATTTGATGGAGTTGGACATGG-30 , antisense: 50 -TGTTCTTGAGTGAAGGACTGAG-30 ; b-catenin: sense: 50 -TAACTCGAGGAGGAGCTGGA-30 , antisense: 50 -GCCAAGGTTGTGAGGTTAGG-30 ; cyclin D1: sense: 50 -TGGAGCCCCTGAAGAAGAG-30 , antisense: 50 -AAGTGCGTTGTGCGGTAGC-30 ; b-actin: sense: 50 -GGCATCGTGATGGACTCCG-30 , antisense: 50 -GCTGGAAGGTGGACAGCGA-30 . Western Blot
TagedPProteins of cultured neurons were extracted according to the manufacturer's instructions (Applygen Technologies Inc., Beijing) and quantified with the Bicinchoninic Acid Protein Assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA). Equal proteins were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with the following antibodies: anti-b-catenin (1:200; Cell Signaling Technology, Danvers, MA, USA), anti-c-myc (1:200; Cell Signaling Technology, Danvers, MA, USA), and anti-cyclin D1 (1:200; Cell Signaling Technology, Danvers, MA, USA). After washing with Tris-buffered saline and 0.1% Tween,
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TagedPthe membranes were incubated with Tris-buffered saline and 0.1% Tween -containing fluorescent-labeled secondary antibodies (IRDye; 800-conjugated goat anti-mouse 1:10,000 dilution; Rockland, Gilbertsville, PA) for 1 hour. The bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). A mouse anti glyceraldehyde-3phosphate dehydrogenase (1:5000; Sigma) was used as an internal control.
Statistical Analysis TagedPData were presented as mean § SEM. One-way analysis of variance followed by Student Newman Keuls (S-N-K) posthoc analysis was performed with SPSS V.17.0. P < 0.05 was considered to be statistically significant.
Results Liraglutide Increases the Viability of Cortical Neurons Damaged by Oxidative Stress TagedPFirst, we established whether liraglutide protected cortical neurons under oxidative stress using CCK-8 assays. Results showed that H2O2 decreased the cell viability by approximately 70.82% compared with the control group; the decrease in cell viability was rescued by liraglutide treatment. Moreover, we observed that liraglutide could increase the viability in a dose-dependent manner (Fig 1,
Figure 1. Liraglutide restores cell viability in primary cultured cortical neurons damaged by oxidative stress. Neurons were pretreated with 100 mM H2O2 for 2 hours, subsequently with the treatment of different concentrations of liraglutide for 24 hours. (A) The cell viability was determined by CCK-8 assay. Each group was done with 6 replicates (n = 6), and the experiment was replicated 3 times. Depicted were mean § S.E.M., *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group represents statistic difference. (B) The cell apoptosis was determined by Hoechst 33342 staining. Bar = 50 mm. Depicted were mean § S.E.M., *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group represents statistic difference. Abbreviation: CCK-8, Cell Counting Kit-8
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TagedPA). The viability of cortical neurons was in 31.16 § 4.09%, 40.09 § 3.82%, 63.61 § 5.11%, 53.22 § 5.72% in 1/10/ 100/1000 nM liraglutide group under oxidative stress, respectively. Results revealed that liraglutide could significantly protect neurons against oxidative stress. Considering the effectiveness, 100 nM liraglutide might be suitable to conduct the remaining experiments. TagedPCell apoptosis was also assessed by Hoechst 33342. As shown in Figure 1B, the percentage of the apoptosis cells in control group, H2O2 group, and H2O2 + liraglutide group was 12.20 § 2.05%, 73.05 § 6.63%, and 43.18 § 4.35%, respectively, which suggested that liraglutide could alleviate the H2O2-induced apoptosis of neurons.
Liraglutide Protects Neuritogenesis Ability of Cortical Neurons Insulted by Oxidative Stress TagedPNext, We explored whether liraglutide promoted neurite outgrowth OD cortical neurons under oxidative stress evaluated by measuring the longest neurite. As shown in Figure 2, neurons exposed to H2O2 showed a significant decrease in neurite outgrowth (22.88 § 2.82 mm), compared with the control group (35.54 § 5.21 mm). However, liraglutide significantly recovered the decrease in neurite outgrowth, (30.30 § 2.97 mm). These results suggested that liraglutide could effectively protect neuritogenesis of neurons subjected to oxidative injury.
A Wnt Pathway Inhibitor Xav939 Inhibits LiraglutideInduced Neurite Elongation Under Oxidative Stress TagedPWe further investigated the underlying molecule mechanism in liraglutide-induced neurite elongation under
TagedP xidative stress. Previous studies have shown the Wnt o pathway has been involved in neurogenesis in the subgranular zone.17 In addition, GLP-1 exerts proliferative and protective effects involving the Wnt signaling pathway.19 Therefore, we hypothesize that the Wnt pathway was involved in liraglutide-induced neurite elongation. To test this hypothesis, the Wnt pathway inhibitor Xav939 was used to block the Wnt pathway. As shown in Figure 2, Xav939 significantly prevented liraglutide-mediated neurite outgrowth of cortical neurons under H2O2. These data indicate that liraglutide restored neurite outgrowth of cortical neurons under oxidative stress partially blocked by Xav939.
The Wnt Pathway is Required for Liraglutide-Induced Neurite Outgrowth Under Oxidative Stress TagedPSince Xav939 could counteract the promoting effect of liraglutide on neurite outgrowth of cortical neurons under oxidative stress, and b-catenin, c-myc, and cyclin D1 were downstream target molecules of the Wnt pathway, we then sought to determine whether b-catenin, c-myc, and cyclin D1 were involved in liraglutide-induced neurite outgrowth. The genes and proteins levels of b-catenin, cmyc, and cyclin D1 were measured using quantitative real-time polymerase chain reaction and Western blot assays. Compared with the control group, the genes and proteins expressions of b-catenin, c-myc, and cyclin D1 were significantly decreased in H2O2 group. However, liraglutide significant increased the b-catenin, c-myc, and cyclin D1 levels. In addition, we also observed Xav939 could prevent the increased b-catenin, c-myc, and cyclin D1 levels that liraglutide induced (Figs 3 and 4). These indicate that the Wnt pathway play an effect in
Figure 2. Xav939 inhibits liraglutide-induced neurite elongation of cortical neurons against oxidative stress. Bar = 20 mm. (A) Representative images of cultured cortical neurons stained with anti-b-tubulin antibody in different treatment. (a) Cont: neurons cultured in medium without H2O2. (b) Neurons cultured with H2O2. (c) Neurons cultured with H2O2 and 100 nM liraglutide. (d) Neurons cultured with H2O2 and 100 nM liraglutide and 0.5 mM Xav939. (B) Statistic results in different groups were shown as above chart. Values represent the mean § S.E.M. (n = 60 neurons). *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference.
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Figure 3. qRT-PCR analysis of the expressions of b-catenin, c-myc, and cyclin D1 mRNAs in different experimental conditions. Liraglutide promotes neurite outgrowth insulted by H2O2 by mediating the Wnt pathway. (A) b-catenin; (B) c-myc; and (C) cyclin D1. Bar graphs with error bars represent mean § SEM. *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference. Abbreviation: qRT-PCR, quantitative real-time polymerase chain reaction.
TagedPliraglutide-mediated neurite outgrowth of cortical neurons under oxidative stress.
Discussion TagedPIn this study, we examined the effect of liraglutide on neurite outgrowth under oxidative stress and the underlying mechanism. To our knowledge, we demonstrated for the first time that liraglutide promoted neuroprotective and neurotropic ability of cortical neurons insulted by oxidative stress partly involved in the Wnt pathway. TagedPBecause diabetes has been identified as a risk factor for disorders of CNS, drug-treated diabetes may be useful to
TagedP ave a neuroprotective effect.23 GLP-1, an intestinalh secreted incretin, can decrease blood sugar by increasing cellular glucose uptake. Recent studies suggest that GLP1 acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, protects synaptic plasticity, and memory formation and reduces plaque formation and the inflammation response.11,12 The emerging data show that GLP-1 and its agonist extendin-4 have neurotrophic function to induce neuronal differentiation of PC12 cells and prevent neurons damage challenged by oxidative stress.24 Moreover, Exendin-4 increased neurite outgrowth of adult sensory neurons in vitro.12 However, liraglutide is a newer GLP-1 analog and also has been
Figure 4. Western blot analysis of the expressions of b-catenin and cyclin D1 proteins in different experimental conditions. (A) Western blot for b-catenin protein levels in each group, (B) bar graph shows the mean § S.E.M. of b-catenin/GAPDH ratio. (C) Western blot for c-myc protein levels in each group, (D) bar graph shows the mean § S.E.M. of c-myc/GAPDH ratio. (E) Western blot for cyclin D1 protein levels in each group, (F) bar graph shows the mean § S.E.M. of cyclin D1/GAPDH ratio. *P < 0.05 versus Cont group, #P < 0.05 versus H2O2 group, $P < 0.05 versus H2O2 + liraglutide group represents statistic difference. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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agedPrT eleased onto the market several years ago.5 It has a longer biological half-life, but also can cross the blood-brain barrier,25,26 which are of vital importance for treat disorders of the CNS. Liraglutide has been found to have neuroprotective action in various experimental models. Liraglutide significantly decreased the infarct volume and improved neurologic deficits in a rat model of middle cerebral artery occlusion.6 Liraglutide exerts protective effects on memory formation, synaptic plasticity, synapse survival, and a reduction of amyloid synthesis and plaque load in the brain.27 Liraglutide significantly increased memory retention and total hippocampal CA1 pyramidal neuron numbers in SAMP8 mice.9 In addition, Li et al reported that liraglutide promoted neurite outgrowth of primary cortical neurons in vitro.10 Here, liraglutide treatment rescued the viability and decreased apoptosis of neurons insulted by H2O2 in vitro model. The neurite outgrowth assay further illustrated liraglutide could exert a neuroprotective effect on neurite outgrowth of cortical neurons against oxidative stress. These findings suggested that liraglutide could block cellular injury and promote neurite outgrowth ability of cortical neurons under oxidative stress. TagedPThe Wnt pathway is important in regulation of many cellular events, including prevention of cellular apoptosis as well as promotion of cell regeneration.28 In brain, the Wnt pathway not only serves to foster neuronal survival,29 but also be associated with axonal remodeling, dendritic morphogenesis, and neuronal connectivity.30-32 The Wnt pathway has been shown to regulate neurite outgrowth by increasing neurite spreading and branching as well as expression of synapsin I, a protein that participates in synapse formation and neurotransmitter release in primary neuronal cell culture.33 Targeted disruption of the Fzd3 gene, one of the Wnt pathway genes, in mouse produced severe defects in several major axon tracts of the forebrain.34 Over-expression of b-catenin has inhibited neurite outgrowth in embryonic mouse retinal explant cultures and PC12 cells.35,36 In addition, a conserved Wnt pathway which is crucial for anterior-posterior outgrowth of neurites from RME head motor neurons in Caenorhabditis elegans important role of a Wnt-Frz/Ror-Dsh pathway in regulating neurite anterior-posterior outgrowth.37 Dishevelled-2/3 mediates Wnt-3a-dependent neurite outgrowth in Ewing sarcoma family tumor cells.38 It is reported that GLP-1 is a direct activator of the Wnt pathway.39 Therefore, the Wnt pathway implicated in the regulation of liraglutide-induced neurite outgrowth could be of great potential to evaluate the underlying mechanism, in our study, treatment with a special Wnt pathway inhibitor Xav939 partially inhibited the enhancing effect played by liraglutide in neurite outgrowth. Consistently, the gene and protein levels of b-catenin, c-myc, and cyclin D1 were significantly increased by the liraglutide treatment, however, partially reduced by Xav939. These data indicate that the Wnt pathway may at least partially contribute to
lTagedP iraglutide-induced neuritogenesis of cortical neurons under H2O2, which may provide a new insight to promote brain plasticity. TagedPIn conclusion, we have shown that liraglutide restored the viability, inhibited apoptosis, and protected neurite outgrowth of cortical neurons under oxidative stress. One of the related mechanisms may be the activating of the Wnt pathway. But more experiments should be carried out to further clarify the exact mechanisms. In addition, it needs to be further determined that whether the conclusion in this study can also apply to animals or clinical trials.
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TagedP30. TagedP31. TagedP32. TagedP33. TagedP34. TagedP35. TagedP36. TagedP37. TagedP38. TagedP39.
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