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Keywords:

  • Alzheimer's disease;
  • apoptosis;
  • incretins;
  • insulin;
  • neurodegeneration;
  • neuroprotection

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References
Thumbnail image of graphical abstract

Glucagon-like peptide 1 (GLP-1) is a growth factor that has demonstrated neuroprotective properties in a range of studies. In an APPswe/PS1ΔE9 mouse model of Alzheimer's disease (AD), we previously found protective effects on memory formation, synaptic plasticity, synapse survival and a reduction of amyloid synthesis and plaque load in the brain. Here, we analyse the neuroprotective properties of the GLP-1 analogue liraglutide in human neuroblastoma cell line SH-SY5Y during methyl glyoxal stress. We show for the first time that cell viability was enhanced by liraglutide (XTT assay) in a dose-dependent way, while cytotoxicity (LDH assay) and apoptosis were reduced. Expression of the pro-survival Mcl1 signaling protein was increased, as was activation of cell survival kinases Akt, MEK1/2 and the transcription factor p90RSK. Liraglutide also decreased pro-apoptotic Bax and Bik expression. In addition, the membrane potential and the influx of calcium into the cell were enhanced by liraglutide. GLP-1 receptor expression was also increased by the drug. The results demonstrate a range of growth factor-related cytoprotective processes induced by liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza®). It is also tested in clinical trials in patients with Alzheimer disease.

We investigated the neuroprotective properties of the GLP-1 analogue liraglutide in SH-SY5Y cells during methyl glyoxal stress. Cell survival was enhanced, while cytotoxicity and apoptosis was reduced. Expression of the pro-survival Mcl1 signaling protein, activation of Akt, MEK1/2 and the transcription factor p90RSK was increased. Pro-apoptotic Bax and Bik expression was reduced, and the influx of calcium into the cell was normalised. The results demonstrate a range of growth factor related cytoprotective processes induced by the drug liraglutide.

Abbreviations used
AD

Alzheimer's disease

BBB

blood–brain barrier

BDNF

brain derived neurotrophic factor

BrdU

5-bromo-2-2deoxyuridine

ERK

extracellular signal-regulated kinase

LDH

lactate dehydrogenase

MG

methyl glyoxal

NGF

nerve growth factor

PBS

phosphate buffered saline

PI3k

phosphoinositide 3 kinase

PI

propidium iodide

PKA

protein kinase A

PKB

protein kinase B

SFM

serum free medium

Glucagon-like peptide 1 (GLP-1) is a signaling peptide that has a range of important physiological properties. Due to the fact that GLP-1 has beneficial effects on insulin release from the pancreas during episodes of hyperglycaemia (Drucker and Nauck 2006), a range of long-lasting GLP-1 mimetics has been developed as treatments for type 2 diabetes. Currently, three such mimetics are on the market and are prescribed to diabetics, exendin-4 (Byeatta®), liraglutide (Victoza®), and lixisenatide (Lyxumia®) (Madsbad et al. 2008; Vilsboll 2009; Christensen et al. 2011). The beneficial effects of these drugs are not limited to the treatment of diabetes. GLP-1 has growth-factor properties (Greig et al. 2004; Holscher 2011) and has been shown to improve cardiovascular conditions (Noyan-Ashraf et al. 2009; Kim et al. 2013). We found in a range of preclinical studies that the GLP-1 analogue liraglutide crosses the blood–brain barrier into the brain and shows a range of neuroprotective effects, eg. protection of memory formation in a mouse model of Alzheimer's disease (AD), protection of synaptic plasticity in the hippocampus, prevention of synapse loss and reduction of amyloid plaque levels and soluble amyloid oligomer levels (McClean et al. 2010, 2011; Hunter and Holscher 2012; Han et al. 2013), normalisation of neurogenesis and progenitor proliferation in the hippocampus (Hamilton et al. 2011; Parthsarathy and Holscher 2013a), and a reduction of the chronic inflammation response in the brain (McClean et al. 2011; Parthsarathy and Holscher 2013b).

In the present study, we are investigating the cellular mechanisms and cell signaling pathways that underlie the cytoprotective and anti-inflammatory effect. While the signaling mechanisms activated by GLP-1 in beta-cells in the pancreas have been investigated in detail (Doyle and Egan 2007), very little is known about the signaling pathways in neurons. Few studies have investigated what second messenger pathways are activated or modulated by GLP-1. Importantly, GLP-1 signaling affects the release of other growth factors. In one in vivo study, peripheral injection of GLP-1 releasing cells increased the levels of mRNA expression of nerve growth factor-inducible protein A in the brain (Fan et al. 2010). GLP-1 also enhanced the release of brain derived neurotrophic factor (BDNF) at the synapse (Mattson 2012). Importantly, insulin signaling is normalised by GLP-1, and peripheral injection of exendin-4 reversed the insulin desensitisation in the brain of APP/PS1 mice (Bomfim et al. 2012). In a cell culture study of PC12 cells, GLP-1 and exendin-4 were found to enhance nerve growth factor (NGF)-induced cell differentiation into neurons (Perry et al. 2002). Activating the GLP-1 receptor in neurons activates an adenylyl cyclase and increases cAMP levels, which in turn activates protein kinase A (PKA) and cyclic AMP response element binding protein (CREB) (Perry and Greig 2004; Hunter and Holscher 2012). Furthermore, neuroprotective effects of GLP-1 in PC12 cells were initiated via the phosphoinositide 3 kinase (PI3k) and Akt/PKB pathway and also involved mammalian target of rapamycin activation (Perry and Greig 2005; Kimura et al. 2009), a growth factor downstream pathway that is also activated by insulin (Nelson and Alkon 2005). The PKA and PI3k pathway was activated and apoptosis via Caspase-3 was reduced in this cell culture study (Li et al. 2010). Other studies confirmed the anti-apoptotic property of GLP-1 mimetics in cultured neurons (During et al. 2003; Perry et al. 2003). In cultured neurons, exendin-4 was neuroprotective in a PKA or PI3k activity dependent way. In peripheral neurons, GLP-1 receptor activation increased extracellular signal-regulated kinase (ERK) signaling and enhanced the total ERK to pERK ratio, comparable to NGF induced ERK signaling (Jolivalt et al. 2011).

Furthermore, cytokine release is reduced by the GLP-1 analogue liraglutide injected ip. in an x-ray inflammation response in mice, (Parthsarathy and Holscher 2013b), and in an in vivo study of APP/PS1 mice, exendin-4 reduced JNK cytokine cell signaling (Bomfim et al. 2012). Ca2+ is an important second messenger in neurons, and abnormal Ca2+ levels are neurotoxic. Beta-amyloid disturbs the Ca2+ homeostasis, which is considered one of the molecular mechanisms of how beta-amyloid kills neurons in AD (Holscher 1998). GLP-1 analogues normalised Ca2+ levels in beta cells stressed with methyl glyoxal (MG) (Selway et al. 2012). We chose this stressor as it compromises energy metabolism in the cell, which is a common observation in neurons in AD (Hoyer 2004; Mattson 2012). In cultured neurons, GLP-1 normalised glutamate-induced voltage gated calcium channel dependent Ca2+ influx into neurons which protected the cells from glutamate induced toxicity (Gilman et al. 2003). We therefore investigated if liraglutide has effects on the Ca2+ levels of human neuroblastoma cells.

To further investigate the neuroprotective effects of the GLP-1 analogue liraglutide, we tested the drug in the human neuroblastoma cell line SH-SY5Y, using MG as a stressor. We evaluated the effects of the drug on cell survival, proliferation, and apoptosis. We investigated the signaling pathways involved in the cytoprotective actions and measured levels of growth factor kinases phosphorylated Akt, MEK1/2 and the transcription factor p90RSK, as well as apoptotic markers caspase-3, Bax and Bik, and survival marker Mcl1. In addition, changes in membrane potential and total intracellular Ca2+ induced by MG stress were measured.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References

Materials

Liraglutide was purchased from GL Biochem (Shanghai) Ltd. The purity of the peptide was analysed by reversed-phase HPLC and characterized using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Cell proliferation kit II (XTT) and cell proliferation ELISA 5-bromo-2-2deoxyuridine (BrdU) (colorimetric) kit were purchased from Roche and the CytoTox 96 Non-Radioactive Cytotoxicity Assay assay kit from Promega (Madison, WI, USA). Image-iT LIVE Green poly caspase detection kit, anti-rabbit goat Alexa555 secondary antibody and phosphate buffered saline (PBS tablets) were purchased from Invitrogen (Paisley, UK). FLIPR Membrane Potential Assay kit and FLIPR calcium assay kit were purchased from Molecular devices (Berkshire, UK). Cleaved caspase-3 (Asp175), Mcl1, Bax, Bik polyclonal and phospho-AKT (Ser 473), phospho-MEK1/2 (Ser217/221), phospho-p90RSK (Ser380) monoclonal, primary antibodies raised in rabbit and horseradish peroxidase labeled secondary antibody, were purchased from Cell signaling technology (Hertfordshire, UK). Millicell EZ 8-well sterile glass slides were purchased from Merck Millipore (Watford, UK). Cell lysis buffer was obtained from Cell signaling technology, Quick start protein assay reagent from Biorad (Dublin, Ireland), X-Ray film, polyvinylidene difluoride membranes and Amersham ECL Prime western blotting detection reagent from GE, Healthcare Lifesciences (Buckinghamshire, UK). Other materials for western blotting and cell culture were obtained from Invitrogen.

Cell culture

The human neuroblastoma cell line SH-SY5Y was obtained from LGC standards (ATCC No. CRL-2266), and maintained in Dulbecco's minimum essential medium, DMEM+F12 (1 : 1) GlutaMax supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 100 Units per ml of Penicillin and 100 μg per mL of Streptomycin at 37°C in a humidified incubator with 95% air and 5% CO2. The cells were sub-cultured when 80–90% confluent and seeded at 1 : 5 ratio. Media was changed every 4–5 days.

Measurement of cell viability, cytotoxicity and proliferation

Cell viability, cytotoxicity, and proliferation were assessed using 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Cell proliferation kit II), Lactate dehydrogenase (LDH, CytoTox 96 assay kit) and 5-bromo-2-deoxyuridine (BrdU, Cell proliferation ELISA colorimetric kit) incorporation, respectively.

The assay was formatted by seeding SH-SY5Y cells in laminin-coated 96-well plates at a density of 5 × 104 cells/well for 24 h. The cells were serum starved for 12 h in serum free medium (SFM) and were pre-treated for 4 h with different concentrations of Liraglutide. Medium was removed and the cells were exposed to various concentrations of MG for 12 h. Supernatant was removed (to be used for LDH assay) and 49 μL of XTT and 1 μL of electron-coupling reagent diluted 1 : 1 with SFM was added to each well and the plate incubated at 37°C for 4 h. XTT measures cell viability based on the activity of mitochondrial enzymes in a live cell that reduces XTT to a highly water-soluble product, λmax 492 nm, which is directly proportional to the number of living cells in the sample. The plate was gently shaken for 5 min on Microtitre plate shaker (Stuart, Staffordshire, UK) and absorbance measured at 492 nm and 690 nm in a FLUOstar Omega microplate reader (BMG LABTECH, Aylesbury, Bucks, UK).

Cell proliferation was assessed in actively proliferating cells by measuring the amount of BrdU incorporated in newly synthesized DNA strands using a BrdU cell proliferation kit. BrdU incorporation was measured as per the manufacturer's instructions. Briefly, after 12 h of MG stress, BrdU labeling reagent was added for 4 h and the plate incubated at 37°C. BrdU incorporation was measured immunochemically by peroxidase labeled anti-BrdU antibody followed by colorimetric substrate detection. The plate was read at 370/492 nm λ using microplate reader. The amount of BrdU incorporated in the proliferating cells was directly proportional to the increasing optical density.

The CytoTox 96 assay kit was used to quantitatively measure the LDH, a stable cytosolic enzyme, which is released upon cell lysis, according to the manufacturer's instructions. LDH in supernatant is measured in an enzymatic assay that results in the formation of a red formazan product and the amount of colour formed is proportional to the number of lysed cells.

Immunocytochemistry

SH-SY5Y cells were grown on laminin coated eight-well chamber slides and pre-treated for 4 h with 100 nM Liraglutide. Medium was removed and the cells were exposed to 600 μM MG for 8 h. Cells were washed with 1X PBS (pH 7.4) and fixed in 4% paraformaldehyde for 10 min followed by permeabilization in 0.3% Triton-X-100 for 5 min. Blocking was done in 1% bovine serum albumin and 5% goat serum for 1 h at 25°C. Cleaved caspase-3 (Asp175) rabbit polyclonal antibody was added at dilution 1 : 400 and incubated at 25°C for 1 h. Cells were then washed three times (5 min each) in 1X PBS followed by incubation at 25°C for 1 h in goat anti-rabbit Alexa555 secondary antibody at dilution 1 : 800, counterstaining with Hoechst 33342 (nuclear stain) for 10 min. Mounting was done in Vectashield mounting medium and microscopy was performed using Axio Scope 1 (Zeiss, Cambridge, UK). Imaging of GLP-1R was performed using a confocal microscope (Leica Microsystems, Milton Keynes, UK; SP5 LAS IF Software).

Apoptotic insult

Apoptotic cells were assessed by differential Hoechst 33342 and propidium iodide (PI) staining. SH-SY5Y cells were grown on laminin coated eight-well chamber slides and pre-treated for 4 h with 100 nM Liraglutide. Medium was removed and the cells were exposed to 600 μM MG for 8 h. Cells were washed with 1X PBS (pH 7.4) and fixed in 4% paraformaldehyde for 10 min. Fixed cells were washed twice with 1X PBS (pH 7.4) followed by addition of 1 μg/ml Hoechst 33342 and PI and incubated at 25°C for 15 min. Mounting was done in Vectashield mounting medium and observed under Fluorescence microscope. Hoechst 33342 stains nuclei blue and shows typical apoptotic features such as chromatin condensation and fragmentation. The intact membrane of the healthy cells excludes PI but necrotic cells are stained brightly as their membrane integrity is compromised. So, the apoptotic cells were quantified by counting the magenta coloured nuclei.

Measurement of total caspase

Image-iT LIVE Green poly caspase detection kit was used to detect active caspases based on a fluorescent inhibitor of caspases, FAM-VAD-FMK poly caspases reagent (FLICA). Cells were grown on laminin-coated eight-well chamber slides and pre-treated for 4 h with 100 nM Liraglutide. Medium was removed and the cells were exposed to 600 μM MG for 8 h and thereafter, 1X FLICA reagent was added in sufficient amount to cover the cells followed by incubation at 37°C for 1 h. After gently removing the solution and gently rinsing the cells with SFM, Hoechst 33342 was used to counterstain the nucleus and mounted in apoptosis fixative solution and observed under a Fluorescence microscope. FLICA reagent has approximate excitation/emission maxima of 488/530 nm and associates a fluoromethyl ketone (FMK) moiety, which can react with a caspase-specific amino acid sequence (valine-alanine-aspartic acid, VAD) and a carboxyfluorescein group (FAM) is attached as a reporter. Thus, green fluorescent signal observed is a direct measure of the total active caspases present.

Measurement of total intracellular calcium and membrane potential

Intracellular calcium and membrane potential were measured using FLIPR calcium assay and Membrane Potential Assay kit. 5 × 104 cells/well were seeded in Laminin pre-coated clear bottom 96 well black plates for 24 h and pre-treated for 4 h with different concentrations of Liraglutide. Cell culture assay medium was removed and the cells were exposed to 300 μM and 600 μM MG for 8 h. Intracellular Ca2+ responses and membrane potential were monitored using FlexStation scanning fluorimeter with integrated fluid transfer workstation (Ojo et al. 2011).

Western blotting

2 × 106 cells were grown in Laminin pre-coated 100 mm plates and total protein was extracted. Cells were washed with cold 1X PBS buffer followed by addition of cell lysis buffer containing protease inhibitors. After two freeze thaw cycles, and incubation at 4°C on a shaking platform, the lysate was collected and total proteins isolated after centrifugation at 20 000 g for 15 min. Quick start protein assay reagent was used to estimate the protein concentration based on the principle of Bradford method that involves binding of Commasie Brilliant Blue G-250 dye to proteins. A 10 mg/mL stock solution of bovine serum albumin was prepared and further diluted to 1.5, 1.0, 0.75, 0.5, 0.25, 0.125, and 0.0625 mg/mL using double distilled water. 5 μL of the diluted sample was added into a 96 well clear bottom plate in quadruplicate along with 20 μL of double distilled water and 150 μL of the Quick start protein assay reagent and the plate read after 5 min at 595 nm.

Cell lysate containing 5 μg of protein was separated on 4–12% gradient Bis-Tris gel with Novex pre-stained marker and electrophoresed in running buffer at 200 mV for 35 min followed by transfer to polyvinylidene difluoride membrane. Following protein transfer, the membrane was washed in 1X TBST (tris-buffered saline with 0.05% Tween-20, pH 8) and blocked in 5% skimmed milk for 1 h at 25°C. The membrane was then incubated with anti-Mcl1 (1 : 200), anti-Bax (1 : 200), anti-Bik (1 : 200), anti-pAkt (Ser473) (1 : 400), anti-phospho-MEK1/2 (Ser217/221) (1 : 1000), anti-phospho-p90RSK (Ser380) (1 : 1000) antibodies at 25°C for 1 h and after three washes (10 min each) in TBST further incubated with 1 : 400 horseradish peroxidase-conjugated anti-rabbit IgG. All the primary antibodies used were generated in rabbit. The protein bands were visualized by Amersham ECL Prime western blotting detection reagent according to the manufacturer's recommendation. Image J was used to perform the densitometric analysis of each band density. Selecting each band on the scan created the profile plot and the peaks corresponded to the dark bands in the original image. GAPDH was used as loading control and relative density of the peaks were calculated in Excel spreadsheet after normalizing with GAPDH. To reprobe, the membranes were incubated in a 0.5 M Tris HCl buffer pH 6.8 containing 10% SDS with 0.8% ß-mercaptoethanol with some agitation at 50°C for 45 min followed by thorough rinse in water for 1 h and extensive washing in TBST for 5 min. The membranes were checked with chemiluminescent detection preceding incubation in another primary antibody of interest.

Statistical analysis

Statistical analysis was done using Prism 5.0 (GraphPad software Inc., La Jolla, CA, USA) with the 95% level of probability and the results were presented as mean ± SEM. Data were analysed by unpaired Student's t-test and one-way anova, followed by Bonferroni's multiple comparison post-hoc.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References

Liraglutide pre-treatment increases cell survival and cell proliferation and reduces cell cytotoxicity

To determine the neuroprotective effect of Liraglutide in the SH-SY5Y human neuroblastoma cells from oxidative stress by Methylglyoxal (MG), we performed the XTT and LDH assays to study cell viability and cytotoxicity, respectively. To examine whether GLP-1 analogue, Liraglutide can protect SH-SY5Y cells from MG toxicity, cells were pre-treated with 10, 100, and 200 nM Liraglutide and after stressed with 300, 600 and 1200 μM MG for 12 h and cell viability determined by XTT assay (Fig. 1a). Liraglutide pre-treatment significantly ameliorated 600 μM MG induced stress at 100 (p < 0.001) and 200 nM (p < 0.01) when compared to 0 nM Liraglutide. Although 1200 μM MG concentration has the highest impact on reducing the cell viability, 200 nM Liraglutide was still effective significantly (p < 0.05). A 50% decrease in cell viability at 600 μM MG was restored to 80% upon pre-treatment with 100 nM Liraglutide. For additional assessment of cell viability, LDH levels were quantified in the supernatant collected after 12 h of MG stress. As shown in Fig. 1b, pre-treatment of SH-SY5Y cells with 100 and 200 nM Liraglutide conferred similar protective effects, almost reversing the increased LDH levels, especially at 600 μM MG stress (p < 0.001). Together, these results demonstrate that 100 nM Liraglutide is protective against 600 μM MG induced stress in SH-SY5Y cells. We considered 600 μM concentration of MG stress for further studies as 100 nM Liraglutide exhibited highly significant effect at this stress, both shown by XTT and LDH assays.

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Figure 1. Neuroprotective effect of glucagon-like peptide 1 analogue, Liraglutide against methyl glyoxal (MG) induced cell death. Liraglutide pre-treatment increase cell survival and cell proliferation and reduces cell cytotoxicity. (a) Liraglutide protected SH-SY5Y cells from 300, 600, and 1200 μM concentration of MG induced stress. Cells were pre-treated with 10, 100, and 200 nM Liraglutide for 4 h followed by exposure to different concentration of MG for 12 h. (b) Liraglutide (100 and 200 nM) pre-treatment prevented MG (300, 600, and 1200 μM) induced increase in levels of lactate dehydrogenase (LDH). (c) Cell proliferation [5-bromo-2-2deoxyuridine (BrdU)]: Increasing concentration of Liraglutide (10, 100, 200 nM) pre-treatmemt for 4 h elevated BrdU concentration after 12 h versus untreated controls. 100 nM Liraglutide dose induced maximum BrdU incorporation followed by 0 and 300 μM MG induced stress. Data are presented as mean ± SEM and as a percentage of control. Statistical analysis was done by one-way anova followed by Bonferroni's Multiple Comparison test (*< 0.05, **p < 0.01, ***< 0.001), n = 5. (a–b) Statistical analyses of 0 μM MG versus 300, 600 and 1200 μM MG without Liraglutide (p < 0.001).

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After showing successful neuroprotective effects by Liraglutide pre-treatment, we next thought to examine the proliferative effects by monitoring BrdU incorporation. A dose dependent increase in cell proliferation was observed after Liraglutide treatment. Figure 1c shows a threefold increase in cell proliferation at 100 nM Liraglutide (p < 0.01) concentration when stressed with 300 μM MG. Interestingly, there was no significant difference in proliferation in cells stressed with 600 μM MG, demonstrating cell protection by Liraglutide.

Liraglutide pre-treatment increases GLP-1R expression

To confirm the presence of GLP-1 receptor on SH-SY5Y cells, we performed an immunostaining with the GLP-1R antibody in cells exposed to 0 nM (Fig. 2a) and 100 nM (Fig. 2b) Liraglutide for 4 h. GLP-1R fluorescence was quantified to compare total cell fluorescence in Liraglutide treated (68881.27 ± SEM) v/s un-treated cells (39034.9 ± SEM) (Fig. 2c). A 1.76 fold increase in the GLP-1R expression was observed (p < 0.01).

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Figure 2. Representative photomicrographs showing GLP-1R immunostaining in SH-SY5Y cells, 40X. (a) 0 nM Liraglutide (Control), (b) 100 nM Liraglutide pre-treatment for 4 h. Nuclei were stained with Hoechst 33342 and imaging performed using a confocal microscope. (c) Quantification of GLP-1R fluorescence by Image J. 100 nM Liraglutide treated SH-SY5Y cells (68881.27 ± SEM) when compared with non-treated cells (39034.9 ± SEM) shows a significant 1.76 fold increase in the corrected total cell fluorescence (CTCF) of GLP-1R expression. Data are presented as mean ± SEM. Statistical evaluation done by Unpaired t-test, **p < 0.01.

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Liraglutide pre-treatment protects against MG induced apoptosis

To elucidate whether the neuroprotection conferred by Liraglutide pre-treatment against MG induced stress involves protection from apoptosis, we performed Hoechst 33342 and PI staining on the cells pre-treated with 100 nM Liraglutide, after stressed for 12 h with 600 μM MG stress. Figure 3d shows a decrease in apoptotic cells in the Liraglutide treated (19.65 ± SEM) as compared to un-treated ones (31.67 ± SEM) (Fig. 3c). Number of apoptotic cells was quantified and a significant decrease (p < 0.001) of 37.9% in the % apoptotic cells was observed (Fig. 3f).

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Figure 3. Liraglutide pre-treatment protects against MG induced apoptosis. Representative images of three independent experiments showing staining of SHSY5Y cells with Hoechst 33342 and propidium iodide (PI), 20X. (a) 0 nM Liraglutide followed by 0 μM MG (Control), (b) 100 nM Liraglutide followed by 0 μM MG, (c) 0 nM Liraglutide followed by 600 μM MG, (d) Pre-treated with 100 nM Liraglutide for 4 h followed by 600 μM MG for 12 h. (e) Representative image showing apoptotic (thick arrow) and normal (thin arrow) nuclei, 100X, (f) Quantification of apoptotic cells. 100 nM Liraglutide pre-treated SHSY5Y cells (19.65 ± SEM) when compared with non-treated 600 μM MG stressed cells (31.67 ± SEM) shows a significant decrease of 37.9% in the%age of apoptotic cells. Data analysed by One-way anova followed by Bonferroni's Multiple Comparison test, ***p < 0.001.

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Liraglutide pre-treatment decreases caspase expression

To confirm the effect of Liraglutide in reducing the apoptotic effect induced by MG, we performed caspase-3 immunostaining in 100 nM Liraglutide pre-treated SH-SY5Y cells stressed with 600 μM MG for 8 h. Figure 4d and e shows a decrease in the expression of cleaved form of caspase-3 in the Liraglutide treated (32966.47 ± SEM) as compared to the un-treated cells (44258.13 ± SEM) (Fig. 4c and e). Corrected total cell fluorescence of caspase-3 expression was measured (Fig. 4e) and a significant decrease of 25.5% was observed.

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Figure 4. Representative photomicrographs showing caspase-3 immunostaining and caspase Live Imaging in SH-SY5Y cells stressed with 600 μM Methylglyoxal for 8 h, 20X. Caspase-3 immunostaining (a) 0 nM Liraglutide followed by 0 μM MG (Control), (b) 100 nM Liraglutide followed by 0 μM MG, (c) 0 nM Liraglutide followed by 600 μM MG, (d) Pre-treated with 100 nM Liraglutide for 4 h followed by 600 μM MG, (e) Quantification of caspase-3 fluorescence by Image J. 100 nM Liraglutide pre-treated SHSY5Y cells (32966.47 ± SEM) when compared with non-treated 600 μM MG stressed cells (44258.13 ± SEM) shows a significant decrease of 25.5% in the corrected total cell fluorescence (CTCF) of caspase expression. Caspase Live Imaging (using Image-iT Live Green Poly Caspases Detection kit) (f) 0 nM Liraglutide followed by 0 μM MG (Control), (g) 100 nM Liraglutide followed by 0 μM MG, (h) 0 nM Liraglutide followed by 600 μM MG, (i) Pre-treated with 100 nM Liraglutide for 4 h followed by 600 μM MG, (j) Quantification of caspase fluorescence by Image J. 100 nM Liraglutide pre-treated SHSY5Y cells (93.2 ± SEM) when compared with non-treated 600 μM MG stressed cells (136.3 ± SEM) shows a significant decrease of 32% in the measured Integrated density of caspase expression. Data analysed by One-way anova followed by Bonferroni's multiple comparison test, **p < 0.01.

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To further corroborate a change in the caspase-3 levels, we performed a polycaspase Live Imaging in 100 nM Liraglutide pre-treated SH-SY5Y cells stressed with 600 μM MG for 8 h. Figure 4i shows a decrease in the expression of caspases in the treated as compared to the un-treated cells (Fig. 4h). Quantification of caspase fluorescence showed a significant decrease of 32% (p < 0.01) in the measured integrated density of caspase expression (Fig. 4j).

Liraglutide pre-treatment decreases pro-apoptotic (Bax and Bik) and increases pro-survival Mcl1 expression

Two other representative markers for apoptotic cell death, Bax and Bik were assessed by western blot analysis. Figure 5a shows altered Bax and Bik expression when cells pre-treated with 100 nM Liraglutide followed by 600 μM MG were compared to untreated cells. Densitometric analysis of Bax (Fig. 5b) and Bik (Fig. 5c) showed a significant decrease (p < 0.001) in the 100 nM Liraglutide treated cells. MG stress resulted in almost a twofold increase in the protein levels of Bax and Bik that was markedly ameliorated by Liraglutide pre-treatment.

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Figure 5. Western blot analysis of pro-apoptotic and pro-survival markers. Analysis of pro-apoptotic markers, Bax and Bik (a). Western blot showing altered Bax and Bik expression when cells were pre-treated with 100 nM Liraglutide followed by 600 μM MG as compared to untreated cells, (b) Densitometric analysis of Bax and (c). Bik. Both the pro-apoptotic markers show a significant decrease. Analysis of pro-survival marker, Mcl1 (d). Western blot showing altered Mcl1 expression when cells were pre-treated with 100 nM Liraglutide followed by 600 μM MG as compared to untreated cells, (e). Densitometric analysis of Mcl1. A significant increase in the Mcl1 expression was observed. Analysis by One-way anova followed by Bonferroni's multiple comparison test, ***p < 0.001.

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In addition to the pro-apoptotic markers, we also performed western blot analysis of Mcl1, a member of pro-survival Bcl-2 family and found a significant decrease (~ 1.8-fold) in its levels in MG stressed cells, which was partially restored to control levels by Liraglutide pre-treatment (Fig. 5d). Densitometric analysis of Mcl1 (Fig. 5e) showed a significant increase (p < 0.001) in the 100 nM Liraglutide treated cells, thus, further confirming an overall decrease in the cell apoptosis as a mechanism involved in neuroprotection conferred by Liraglutide.

Liraglutide pre-treatment restores membrane potential and normalises intracellular calcium levels

The FLIPR membrane potential assay kit was used to detect ion channel modulation by varying the fluorescent signal in accordance to changes in cellular membrane potential. 600 μM MG-induced membrane depolarization was restored (p < 0.01) by Liraglutide pre-treatment in SH-SY5Y cells (Fig. 6a). Figure 6b shows a significant increase (p < 0.001) of intracellular calcium (AUC is area under the curve) in 100 nM Liraglutide pre-treated SH-SY5Y cells stressed with 600 μM MG for 8 h and Fig. 6c shows an increase in relative fluorescence intensity (RFU) of total intracellular calcium.

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Figure 6. Liraglutide pre-treatment restores membrane potential and increases intracellular calcium levels. (a) Membrane potential was measured using FLIPR Membrane Potential Assay kit, (b) Intracellular calcium was measured using FLIPR calcium assay kit (AUC is area under the curve), (c) Relative fluorescence intensity (RFU) of total intracellular calcium. Analysis was done by unpaired t-test (**p < 0.01, ***p < 0.001), n = 6.

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Liraglutide pre-treatment increases pAkt (Ser473), p-MEK1/2 (Ser217/221) and p-p90RSK (Ser380) levels

To further elucidate the mechanism underlying the neuro-protection showed by Liraglutide so far, we conducted western blot analysis of pAkt (Ser473), p-MEK1/2 (Ser217/221) and p-p90RSK (Ser380) levels (Fig. 7a) in the SH-SY5Y cells and found a significant increase (p < 0.01) in the expression of pAkt (Ser473) (Fig. 7b), p-MEK1/2 (Ser217/221) (Fig. 7c) and p-p90RSK (Ser380) (Fig. 7d) in the cells pre-treated with 100 nM Liraglutide stressed with 600 μM MG, compared to the untreated cells.

image

Figure 7. Western blot analysis of pAkt (Ser473), p-MEK1/2, p-p90RSK. (a) Western blot showing altered pAkt (Ser473), p-MEK1/2 (Ser217/221) and p-p90RSK (Ser380) expression when cells were pre-treated with 100 nM Liraglutide followed by 600 μM MG as compared to untreated cells, (b–d) Densitometric analysis of pAkt (Ser473), p-MEK1/2 (Ser217/221), and p-p90RSK (Ser380), respectively, shows a significant increase in their expression levels as analysed by One-way anova followed by Bonferroni's multiple comparison test, **p < 0.01. (e) Diagrammatic representation of the potential pathways that underlies the neuroprotective effects of Liraglutide, mediated by PKB/Akt and MAPK/ERK pathways. Liraglutide stimulates GLP-1R resulting in an increase in the cAMP further leading to intracellular events such as cell survival, inhibition of apoptosis, activation of Ca2+ channels, cell growth, repair and regeneration and regulation of translation/transcription in response to stress. GLP-1R, GLP-1 receptor; PKA, protein kinase A; PI3K, phosphoinositide 3 kinase; PKB, protein kinase B; AC, adenylate cylase; EPAC, exchange proteins directly activated by cAMP; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; CREB, cyclic AMP response element binding protein; P90RSK, ribosomal S6 kinase; MEK1/2, MAPK or Erk kinases; c-Raf, cellular Raf gene (Rapidly accelerated fibrosarcoma); Mcl1, myeloid cell leukaemia protein-1; Casp-9, caspase 9; Casp-3, caspase 3; Bax, Bcl2 associated X protein; Bik, Bcl2-interacting killer; Ca2+, calcium ions.

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Figure 7e demonstrates possible signaling pathways involved in the protective mechanism of the Liraglutide. Increased cAMP activates MEK1/2 (as shown in Fig. 7c) in turn phosphorylating MAPK/ERK resulting in the increased expression of p90RSK (as shown in Fig. 7d), a transcription factor that enters the nucleus and regulates the transcription of genes in response to stress. In addition, GLP-1R activation via PI3K induces PKB/Akt (as shown in Fig. 7b) that is responsible for inhibition of apoptosis and cell survival effects shown by Liraglutide. Akt activation leads to the increased expression of Mcl1 (as shown in Fig. 5e) and reduced expression of apoptotic markers like Bax (as shown in Fig. 5b) and Bik (as shown in Fig. 5c) as well as a decrease in caspase 3 expression (as shown in Fig. 4e and j). Taken together, stimulation of GLP-1R results in an increase in cAMP levels, leading to the intracellular events mediated by PKB/Akt and MAPK/ERK pathways, such as cell survival, inhibition of apoptosis, activation of Ca2+ channels, cell growth, repair and regeneration and regulation of translation/transcription in response to the stress.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References

The results demonstrate that liraglutide, a drug that is on the market as a treatment for type 2 diabetes (Victoza®) (Vilsboll 2009), has neuroprotective properties in human neuroblastoma SH-SY5Y cells. There was a reduction in the methylglyoxal-induced toxicity. Methylglyoxal (MG), a metabolic by-product of glycolysis and has serious toxicological effects when in excess. It interferes with cell energy metabolism, increases oxidative stress and enhances the formation of advanced glycation end products (AGE) (van Eupen et al. 2013; Kaur et al. 2013). As the energy metabolism and glucose utilisation is compromised in the brain of Alzheimer patients (Hoyer 2004; Mattson 2012), the MG stress is a suitable tool to investigate the protective effects of GLP-1 analogues. Insulin desensitisation also interferes with cell energy metabolism (Engelbrecht et al. 2013) and MG induces similar effects (Crisostomo et al. 2013; Engelbrecht et al. 2013). Insulin desensitisation has been described in brains of patients with Alzheimer's disease (Talbot et al. 2012). Therefore, MG induced stress may copy the stressors that neurons are exposed to in this disease.

SH-SY5Y cell numbers treated with high doses of MG were much reduced. For the first time, we show that liraglutide protected cells from MG toxicity and increased the cell proliferation and pro-survival Mcl1 signaling protein, which is a member of the Bcl-2 family (Vlacic-Zischke et al. 2011). Activation of the growth factor signal and cell survival kinases Akt and MEK1/2 was also increased by liraglutide. These kinases activate the transcription factor p90RSK. In addition, liraglutide reduced the induction of apoptosis and caspase and pro-apoptotic Bax and Bik expression (see summary Fig. 7e). Also, the membrane potential and the influx of Ca2+ into the cell was enhanced by liraglutide treatment. In neurons, Ca2+ influx after the initiation of action potentials is an important intracellular messenger for the activation of key enzymes and the modulation of synaptic activity and signal transduction (Bliss and Collingridge 1993). Dysregulation of Ca2+ signaling and of the membrane potential can severely interfere with neuronal firing, synaptic plasticity and signal processing (Stanton 1996). The normalisation of Ca2+ levels in neurons is a major factor in protecting neurons and preserving their functionality. As beta-amyloid is known to destabilise Ca2+ levels in neurons (Holscher 1998), GLP-1 mimetics may be protective as well. Previous research found that GLP-1 activates L-type voltage dependent calcium channels and thereby activates ERK growth factor signaling (Kennedy 1989; Selway et al. 2012). It is of interest to note that GLP-1 receptor expression was increased after treatment with liraglutide, thereby enhancing GLP-1 signaling and potentiating the effects of liraglutide over time. Treatment of the cells with liraglutide furthermore increased the expression of GLP-1 receptors, as had been reported in a previous study that showed an increase in GLP-1R expression in vivo after drug treatment (Romani-Perez et al. 2013). This increase of expression suggests a positive feedback loop to enhance GLP-1 signaling.

The activation of growth factor signaling and the reduction of apoptosis is most likely the underlying molecular mechanism for the neuroprotective effects of liraglutide found in several studies. An additional property of growth factors is that they reduce inflammation. We have shown that liraglutide reduces a chronic inflammation response (Parthsarathy and Holscher 2013b), which also contributes to the neuroprotective effects. Other growth factors have shown a very similar profile of activating cell growth genes, energy utilization, synapse protection, and synaptogenesis, and a protection of memory formation (Holscher 2011). BDNF has been shown to protect synapses in mouse models of AD. Injecting BDNF icv improved memory formation, reduced the observed impairments of synaptic plasticity in this mouse model, and increased synaptic numbers (Blurton-Jones et al. 2009). Enhancing the BDNF levels in the brain by gene delivery vectors also has demonstrated neuroprotective effects on synapses. The elevation of BDNF levels reversed synapse loss, improves synaptic plasticity and restores learning abilities of a mouse model of AD (Nagahara et al. 2009; Poon et al. 2009). BDNF also activates growth factor cell signaling such as the PI3K and PKB/Akt pathway (Foulstone et al. 1999; Duan et al. 2012). This suggests that growth factors activate similar pathways and can induce similar physiological changes. However, BDNF does not cross the blood–brain barrier (BBB), and therefore it would need to be injected into the brain in order to be effective (Schulte-Herbruggen et al. 2007; Zuccato and Cattaneo 2009). There is also an interaction between growth factors. Recent studies have shown that peripheral injection of GLP-1 mimetics enhance BDNF levels in the brain (Mattson 2012). This is an additional pathway of how GLP-1 can enhance growth factor cell signaling in neurons. A different growth factor that has very similar properties is NGF. NGF was found to protect synapses, synaptic plasticity, and learning abilities in AD mouse models or in nonprimate monkeys (Clarris et al. 1994; Kordower et al. 1997; Covaceuszach et al. 2009). However, NGF does not cross the BBB either, which severely limits the potential use of this growth factor to protect the brain (Bradbury 2005; Heese et al. 2006; Schulte-Herbruggen et al. 2007; Covaceuszach et al. 2009). Therefore, enhancing the GLP-1 signal with mimetics that can cross the BBB may compensate for IGF-1 and Insulin desensitisation, which has been observed in the brains of AD patients (Hoyer 1998; de la Monte 2011; Talbot et al. 2012). Phosphorylation of the insulin receptor ß chain was reduced at positions IRβ pY1150/1151 and IRβ pY960, while the insulin receptor substrate 1 (IRS-1) was hyperphosphorylated at positions IRS-1 pS616 and IRS-1 pS636, which deactivates IRS-1 signaling, and IRS-1 binding to PI3K p85α was also much reduced, reducing the activation of PI3K (Talbot et al. 2012). A histological study of brain tissue of AD patients found similar effects (Moloney et al. 2010). Insulin signaling impairment has detrimental effects on cognition in AD patients (Carro and Torres-Aleman 2004; Craft 2005; Reger et al. 2008). Based on these findings, a phase II clinical trial testing nasal insulin application in patients with AD had been conducted and showed improvement in key biomarkers and cognition (Craft et al. 2012; Freiherr et al. 2013).

The same profile of insulin desensitisation found in the brains of AD patients was also found in the APP/PS1 mouse model of AD (Bomfim et al. 2012), and treatment with the GLP-1 mimetic exendin-4 (Bomfim et al. 2012) or with liraglutide (Wang et al. 2012) reversed the insulin desensitisation profile of insulin cell signaling proteins. In addition, we have shown in previous studies that liraglutide enhances synaptic plasticity in the hippocampus of the rat (McClean et al. 2010; Han et al. 2013), progenitor cell proliferation and neurogenesis in the dentate gyrus in the mouse brain (Hamilton et al. 2011) and reduced amyloid plaque load, amyloid oligomer levels, activated microglia chronic inflammation in the brains of APP/PS1 mice, while preserving spatial learning, synapse numbers in the cortex and hippocampus, and enhancing synaptic plasticity and normalising neurogenesis in the hippocampus (McClean et al. 2011). Based on these encouraging preclinical findings, a clinical trial of liraglutide in patients with AD has started (see http://clinicaltrials.gov/ct2/show/NCT01843075?term=liraglutide+and+alzheimer&rank=1.

The data presented in this study on the neuroprotective effects in SH-SY5Y cells add important information on the cellular mechanisms that underlie the neuroprotective effects observed in the previous studies (Holscher 2012).

Acknowledgement/conflict of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References

The work has been supported by Vice Chancellor's Research Scholarship, University of Ulster, NI, UK and Alzheimer's Research UK. The authors MKS and JJ do not declare a conflict of interest. CH is a named inventor on a patent application by Ulster University that lists GLP-1 analogues as a potential treatment for neurodegenerative diseases.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement/conflict of interest
  7. References
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