- Email: [email protected]
tau mouse model of Alzheimer's disease
Biochemical and Biophysical Research Communications xxx (2017) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease Hong-Yan Cai a, 1, Jun-Ting Yang b, 1, Zhao-Jun Wang b, Jun Zhang b, Wei Yang b, Mei-Na Wu b, *, Jin-Shun Qi b, ** a b
Department of Microbiology and Immunology, Shanxi Medical University, 56 Xinjian South Road, Taiyuan, Shanxi Province, 030001, China Department of Physiology, Shanxi Medical University, 56 Xinjian South Road, Taiyuan, Shanxi Province, 030001, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2017 Accepted 18 November 2017 Available online xxx
Type 2 diabetes mellitus (T2DM) has been identified as a high risk factor for Alzheimer's disease (AD). The impairment of insulin signaling has been found in AD brain. Glucagon-like peptide-1 (GLP-1) is an incretin hormone, normalises insulin signaling and acts as a neuroprotective growth factor. We have previously shown that the long-lasting GLP-1 receptor (GLP-1R) agonist lixisenatide plays an important role in memory formation, synaptic plasticity and cell proliferation of rats. In the follow-up study, we analysed the neuroprotective effect and mechanism of lixisenatide, injected for 60 days at 10 nmol/kg i.p. once daily in APP/PS1/tau female mice and C57BL/6J female mice (as control) aged 12 month. The results showed that lixisenatide could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation in the hippocampi of 12-month-old APP/PS1/tau female mice; activation of PKA-CREB signaling pathway and inhibition of p38-MAPK might be the important mechanisms in the neuroprotective function of lixisenatide. The study demonstrated that GLP-1R agonists such as lixisenatide might have the potential to be developed as a novel therapy for AD. © 2017 Elsevier Inc. All rights reserved.
Keywords: Lixisenatide Alzheimer's disease Amyloid plaques Neurofibrillary tangles Neuroinflammation
1. Introduction Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder, characterized by memory loss and cognitive decline [1]. The pathological features of AD include amyloid plaques, neurofibrillary tangles and neuroinflammation, which are caused by deposits of b-amyloid (Ab) fragments, hyperphosphorylated tau protein and excessive activation of microglia
Abbreviations: T2DM, Type 2 diabetes mellitus; AD, Alzheimer's disease; GLP-1, Glucagon-like peptide-1; GLP-1R, GLP-1 receptor; Ab, b-amyloid; APP, amyloid precursor protein; DPP-4, dipeptidyl peptidase 4; BBB, bloodebrain barrier; APP/ PS1/tau, amyloid precursor protein/presenilin-1/tau protein; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PKA, protein kinase A; CREB, cAMP response element-binding protein; p38-MAPK, p38-Mitogen activated protein kinase; NSAIDs, nonsteroidal anti-inflammatory drugs. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H.-Y. Cai), [email protected] (J.-T. Yang), [email protected] (Z.-J. Wang), [email protected] (J. Zhang), vividmail@ 163.com (W. Yang), [email protected] (M.-N. Wu), [email protected] (J.-S. Qi). 1 With the same contribution.
and astrocyte, respectively [2]. Ab is cleaved from amyloid precursor protein (APP) by b- and g-secretases [3] and is thought to play a central role in the onset and progression of AD, which has led to the amyloid cascade hypothesis [4]. Tau is the axonal protein that normally stabilizes microtubules. Abnormal phosphorylation, misfolding and aggregation of tau lead to neurofibrillary tangle formation and ultimately to neuronal cell death [5]. Activated microglia can lead to neuron damage or death, which can induce further activation of microglia, thus resulting in a self-propagating and detrimental cycle of neuroinflammation and neurodegeneration [6]. Up to now, AD is a huge global burden, because of the lack of effective treatments [7]. Hence, it is imperative to search for new drugs for AD. Epidemiological studies have recently discovered that Type 2 diabetes mellitus (T2DM) is considered as a high-risk factor of AD [8]; T2DM and AD share many clinical and pathological features, one of which is impaired insulin signaling [9]; which opens up the opportunities for developing new treatments of AD. Glucagon-like peptide-1 (GLP-1) is an endogenous 30-amino acid peptide hormone, derived from the processing of proglucagon in intestinal L
https://doi.org/10.1016/j.bbrc.2017.11.114 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
2
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
cells and acts as an incretin to release insulin from pancreatic bcells [10]. It activates a parallel signaling pathway to insulin [11] and does not affect the blood glucose level in normoglycemic people [12,13]. Emerging evidence indicates that GLP-1 acts as a growth factor and has been shown to enhance neurite outgrowth [14]; GLP-1 prevents the release of the inflammatory cytokine IL-1b [15]. Moreover, GLP-1 receptor (GLP-1R) is widely expressed in many regions of CNS, particularly the hippocampus [16]. GLP-1R plays an important role in regulating neuronal plasticity and cell survival. Mice overexpressing GLP-1R in the hippocampus developed increased neurite growth and showed improved learning [17]. However, natural GLP-1 has a half-life of a few minutes as it is broken down by dipeptidyl peptidase 4 (DPP-4) [18]. Thus, it is unsuitable for routine clinical use on account of its short half-life. Lixisenatide, a novel long-lasting GLP-1R agonist, is effective for the treatment of T2DM. It comprises 44 amino acids and is similar in structure to exendin-4 (another GLP-1R agonist), differing by absence of a C-terminal proline at position 38 and addition of six Cterminal lysine residues at position 39. These structural modifications result in the significantly higher affinity for GLP-1R [19]. Importantly, it can cross the bloodebrain barrier (BBB) [20]. Our pervious study showed that lixisenatide rescued spatial learning and memory deficits and hippocampal LTP suppression of rats [21]; lixisenatide effectively inhibited Ab25-35-induced Ca2þ elevation and cytotoxicity in the hippocampal formation cultures [22]. Futhermore, the neuroprotective effect of lixisenatide on AD transgenic mice would be discussed. Therefore, in this study, using immunohistochemical staining and Western blotting technique, we observed whether chronic and systemic administration of lixisenatide could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation in the amyloid precursor protein/presenilin-1/tau protein (APP/PS1/tau) female mouse model of AD at 12 month of age; explored the possible molecular mechanisms. 2. Materials and methods 2.1. Animals Triple amyloid precursor protein/presenilin-1/tau protein (APP/ PS1/tau) AD transgenic mice (MMRRC Stock No: 34830-JAX/3xTgAD), which were obtained from Jackson Laboratory (Bar Harbor, Maine, USA) [23] and then bred in the laboratory animal breeding room, over-expressed mutant APP (APPSWE), PS1 (PS1M146V) and tau (tauP301L). The mutant mouse significantly exhibited amyloid plaques and neurofibrillary tangles between 12 and 15 months [24]. It provided the ideal setting for investigating AD-related neurodegenerative events and showed pathological changes that matched the temporal- and region-specific profiles of what occurred in the brain of AD patients [25]. Because female was related to more serious pathology in AD mice [26,27], APP/PS1/tau female mice from the offspring, were used for the experiments, as well as agematched C57BL/6J (C57) female mice, which were wild-type (WT) as control and were obtained from the experimental animal center in Shanxi Medical University. Mice were maintained on a 12/12 lightedark cycle at temperature 23 ± 1 C in a relative humidity of 60% room. Food and water were available ad libitum. All mice handling and procedures accorded with guidelines of the Shanxi Animal Research Ethics Committee. 2.2. Drug treatment APP/PS1/tau female mice and WT female mice (12-month-old) were randomly assigned to four groups: WT þ saline, APP/PS1/ tau þ saline, WT þ lixisenatide, APP/PS1/tau þ lixisenatide.
Lixisenatide (Sigma, St. Louis, MO, USA), with the purity >99%, analysed by reversed-phase HPLC, was stored in dry form and dissolved in saline (0.9% NaCl) before experiments. In preclinical tests of lixisenatide, the effective dose was 10 nmol/kg bw for lixisenatide [28]. We therefore chose the dose 10 nmol/kg for lixisenatide in order to compare the drug on a similar level. APP/PS1/ tau female mice and WT female mice were injected i.p. with saline or lixisenatide at 10 nmol/kg body weight once daily for 60 days [29]. 2.3. Immunohistochemical staining The mice were sacrificed with heart perfusion fixation with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). Whole-brain tissue was dissected and postfixed within 4% PFA for 24 h. Some of the tissue was dehydrated in 30% sucrose solution and embedded with frozen embedding medium O.C.T. Compound (SAKURA, USA). The brain blocks were sliced into 25-mm-thick coronal sections with the freezing microtome (Leica, CM1850, German). Then, the sections were incubated with 5% hydrogen peroxide at room temperature for 15min and blocked with 5% goat serum (Solarbio, China) for 30min. Brain slices were incubated with the primary antibody overnight at 4 C, and followed by the secondary antibody at 37 C for 2 h. The following primary antibodies were used: mouse monoclonal anti-Ab antibody (dilution 1:500, 803015, Biolegend, USA) and rabbit polyclonal anti-Iba-1 antibody (dilution 1:200, 019-19741, WAKO, Japan). The following secondary antibodies were used: Peroxidase-Conjugated AffiniPure Goat antiRabbit IgG (H þ L) (dilution 1:200, ZB-2301, ZSGB-BIO, China), Peroxidase-Conjugated AffiniPure Goat anti-Mouse IgG (H þ L) (dilution 1:200, ZB-2305, ZSGB-BIO, China). The DAB method was applied for positive area coloration. Some brain tissue was dehydrated in 30% sucrose solution and embedded with paraffin. Brain sections (thickness 2 mm) were prepared with the Leica microtome. Then, the sections were incubated with 3% hydrogen peroxide at room temperature for 15min. Next, they were heated in the micro-oven for 3min with pH 6.0 citrate acid buffer. Sections were washed with PBS (0.5% TritonX100) three times (15min per time) and blocked with 5% goat serum for 30min. Primary antibody, rabbit monoclonal anti-tau (phospho T231) antibody (dilution 1:200, ab151559, Abcam, UK) was incubated at 37 C for 1 h. Secondary antibody, PeroxidaseConjugated AffiniPure Goat anti-Rabbit IgG (H þ L) (dilution 1:200, ZSGB-BIO, China) was incubated at 37 C for 20min. The DAB method was applied for positive cell coloration and positive area coloration. All sections were photographed under the microscope and analysis of percentage stained area and percentage positive cells in the CA1 region of the hippocampus was conducted for amyloid plaques, neurofibrillary tangles and inflammation. The percentage stained area and percentage positive cells of each image (2 images per section, approximately 3-4 sections per mouse, n ¼ 6 mice per group) were quantified with Image-Pro Plus 6.0 (Media Cybernetics, USA). 2.4. Western blotting The hippocampi of mice were dissected. Protein of these tissues was extracted (Tissue Protein Extraction Reagent by Boster, China) and supplemented with complete protease inhibitor (Boster, China). The protein concentration was measured using a bicinchoninic acid protein assay kit after removing debris by low-speed centrifugation. 30 mg sample protein was separated on 12% SDSepolyacrylamide gels. After electrophoresis, protein was transferred onto PVDF membrane and nonspecific binding was
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membrane was incubated with the primary antibody overnight at 4 C, followed by the secondary antibody at room temperature for 2 h. The following primary antibodies were used: rabbit monoclonal anti-phospho-PKA antibody (dilution 1:1000, #5661, Cell Signaling Technology, USA), rabbit monoclonal anti-PKA antibody (dilution 1:1000, #5842, Cell Signaling Technology, USA); rabbit monoclonal anti-phospho-CREB antibody (dilution 1:2500, ab32096, Abcam, UK), rabbit monoclonal antiCREB antibody (dilution 1:1000, ab32515, Abcam, UK); rabbit monoclonal anti-phospho-p38 antibody (dilution 1:1000, #4511, Cell Signaling Technology, USA), rabbit monoclonal anti-p38 antibody (dilution 1:1000, #2387, Cell Signaling Technology, USA). Mouse monoclonal anti-GAPDH antibody (dilution 1:1000, TA-08, ZSGB-BIO, China) was used as loading control. The secondary antibodies were Peroxidase-Conjugated AffiniPure Goat anti-Rabbit IgG (H þ L) (dilution 1:5000, ZB-2301, ZSGB-BIO, China) and Peroxidase-Conjugated AffiniPure Goat anti-Mouse IgG (H þ L) (dilution 1:5000, ZB-2305, ZSGB-BIO, China). The membrane was rinsed with TBST and the immunocomplex was visualized by using an enhanced chemiluminescence detection kit (Beyotime, China). The signals of the membrane were scanned with the FluorChem Scanner and quantified with the Alpha View SA software. 2.5. Statistics All values were displayed as mean ± standard error (SEM). The SPSS 13.0 and SigmaPlot 11.0 statistical packages were used for statistical analyses. The data were examined by a two-way repeated measures analysis of variance (ANOVA). The statistical significance level was set at p < 0.05.
3
3. Results 3.1. Effects of lixisenatide on amyloid plaque load in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on amyloid plaque load in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 7.846, P ¼ 0.041; lixisenatide: F (1,36) ¼ 8.293, P ¼ 0.037; APP/PS1/tau by lixisenatide: F (1,36) ¼ 7.541, P ¼ 0.043). As shown in Fig. 1, Tukey's post hoc tests showed that compared with WT þ saline mice (1.90% ± 0.20%), percentage of Ab-area in the hippocampi of APP/PS1/tau þ saline mice (9.48% ± 1.20%) was increased (P ¼ 0.039); there was no difference in WT þ saline mice and WT þ lixisenatide mice (2.06% ± 0.30%); lixisenatide significantly reduced percentage of Ab-area in the hippocampi of APP/PS1/tau mice (5.87% ± 0.70%), compared to APP/PS1/tau þ saline mice (P ¼ 0.04). 3.2. Effects of lixisenatide on neurofibrillary tangles in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on p-tau positive cell numbers in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 8.984, P ¼ 0.033; lixisenatide: F (1,36) ¼ 8.192, P ¼ 0.038; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.346, P ¼ 0.031). As shown in Fig. 2A and B, Tukey's post hoc tests showed that compared with WT þ saline mice (51.60% ± 4.30%), percentage of p-tau positive cell numbers in the hippocampi of APP/PS1/tau þ saline mice (89.20% ± 6.20%) was
Fig. 1. Lixisenatide reduced amyloid plaques in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of amyloid plaques in four groups. B, Histograms showed that Ab-area of APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; Ab-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
4
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 2. Lixisenatide reduced neurofibrillary tangles in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of neurofibrillary tangles in four groups. B, Histograms showed that p-tau positive cell numbers of APP/PS1/tau þ saline group were increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; p-tau positive cell numbers of APP/PS1/ tau þ lixisenatide group were decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). C, Histograms showed that p-tau-area of APP/ PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; p-tau-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
increased (P ¼ 0.034); there was no difference in WT þ saline mice and WT þ lixisenatide mice (53.20% ± 3.10%); lixisenatide significantly decreased percentage of p-tau positive cell numbers in the hippocampi of APP/PS1/tau mice (62.30% ± 4.90%), compared to APP/PS1/tau þ saline mice (P ¼ 0.037). Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on p-tau-area in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/ PS1/tau: F (1,36) ¼ 7.431, P ¼ 0.044; lixisenatide: F (1,36) ¼ 6.378, P ¼ 0.049; APP/PS1/tau by lixisenatide: F (1,36) ¼ 8.004, P ¼ 0.039). As shown in Fig. 2 A and C, Tukey's post hoc tests showed that compared with WT þ saline mice (6.56% ± 0.60%), percentage of ptau-area in the hippocampi of APP/PS1/tau þ saline mice (32.58% ± 3.90%) was increased (P ¼ 0.045); there was no difference in WT þ saline mice and WT þ lixisenatide mice (6.62% ± 0.70%); lixisenatide significantly decreased percentage of p-tau-area in the hippocampi of APP/PS1/tau mice (14.99% ± 1.40%), compared to APP/PS1/tau þ saline mice (P ¼ 0.043). Lixisenatide could reduce neurofibrillary tangles in the hippocampi of APP/PS1/tau female mice.
3.3. Effects of lixisenatide on neuroinflammation in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on activation of microglia in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 9.981, P ¼ 0.027; lixisenatide: F (1,36) ¼ 9.491, P ¼ 0.029; APP/PS1/tau by lixisenatide: F (1,36) ¼ 7.920, P ¼ 0.039). As shown in Fig. 3, Tukey's post hoc tests
showed that compared with WT þ saline mice (3.67% ± 0.60%), percentage of Iba-1-area in the hippocampi of APP/PS1/tau þ saline mice (16.85% ± 1.90%) was increased (P ¼ 0.033); there was no difference in WT þ saline mice and WT þ lixisenatide mice (4.17% ± 0.60%); lixisenatide significantly decreased percentage of Iba-1-area in the hippocampi of APP/PS1/tau mice (7.41% ± 0.90%), compared to APP/PS1/tau þ saline mice (P ¼ 0.037). Lixisenatide could reduce neuroinflammation in the hippocampi of APP/PS1/tau female mice.
3.4. Lixisenatide relieved the suppression of PKA-CREB signaling pathway in 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pPKA (APP/PS1/tau: F (1,36) ¼ 6.984, P ¼ 0.046; lixisenatide: F (1,36) ¼ 8.492, P ¼ 0.035; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.398, P ¼ 0.03). As shown in Fig. 4 A and D, Tukey's post hoc tests showed that compared with WT þ saline mice (71.34% ± 7.90%), the level of p-PKA in the hippocampi of APP/PS1/ tau þ saline mice (28.52% ± 3.50%) was decreased (P ¼ 0.042); there was no difference in WT þ saline mice and WT þ lixisenatide mice (69.16% ± 7.10%); lixisenatide significantly reversed the low level of p-PKA in the hippocampi of APP/PS1/tau mice (57.07% ± 6.30%), compared to APP/PS1/tau þ saline mice (P ¼ 0.039). Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pCREB (APP/PS1/tau: F (1,36) ¼ 7.164, P ¼ 0.045; lixisenatide: F (1,36) ¼ 9.458, P ¼ 0.03; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.344, P ¼ 0.031). As shown in Fig. 4 B and E, Tukey's post
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
5
Fig. 3. Lixisenatide decreased activation of microglia in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of activation of microglia in four groups. B, Histograms showed that Iba-1-area of APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; Iba-1-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
Fig. 4. Lixisenatide reversed the suppression of PKA-CREB signaling pathway and inhibited the activation of p38-MAPK in APP/PS1/tau female mice. A, B and C, The expression of p-PKA, PKA, p-CREB, CREB, p-p38 and p38 in four groups. D and E, Histograms showed that levels of p-PKA and p-CREB in APP/PS1/tau þ saline group were decreased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; levels of p-PKA and p-CREB in APP/ PS1/tau þ lixisenatide group were increased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group). F, Histograms showed that the level of p-p38 in APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; the level of p-p38 in APP/PS1/ tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group). *P < 0.05.
hoc tests showed that compared with WT þ saline mice (59.30% ± 6.80%), the level of p-CREB in the hippocampi of APP/PS1/
tau þ saline mice (22.53% ± 4.50%) was decreased (P ¼ 0.036); there was no difference in WT þ saline mice and WT þ lixisenatide mice
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
6
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
(58.70% ± 7.10%); lixisenatide significantly relieved the decrease level of p-CREB in the hippocampi of APP/PS1/tau mice (42.70% ± 3.50%), compared to APP/PS1/tau þ saline mice (P ¼ 0.042). 3.5. Lixisenatide inhibited the activation of p38-MAPK in 12month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pp38 (APP/PS1/tau: F (1,36) ¼ 7.904, P ¼ 0.04; lixisenatide: F (1,36) ¼ 7.452, P ¼ 0.044; APP/PS1/tau by lixisenatide: F (1,36) ¼ 8.358, P ¼ 0.036). As shown in Fig. 4C and F, Tukey's post hoc tests showed that compared with WT þ saline mice (11.30% ± 2.50%), the level of p-p38 in the hippocampi of APP/PS1/ tau þ saline mice (53.11% ± 3.90%) was increased (P ¼ 0.043); there was no difference in WT þ saline mice and WT þ lixisenatide mice (12.30% ± 2.70%); lixisenatide significantly inhibited the increase level of p-p38 in the hippocampi of APP/PS1/tau mice (36.20% ± 3.30%), compared to APP/PS1/tau þ saline mice (P ¼ 0.038). 4. Discussion GLP-1R agonists could be taken by non-diabetic people as they only had the effect on the blood sugar level in hyperglycaemia [20]. The study showed that body weight, plasma glucose, plasma insulin and hippocampal insulin in the AD mouse model, were not affected by liraglutide (another long-lasting GLP-1R agonist) treatment [30]. So, lixisenatide also did not promote insulin secretion when the plasma glucose level was in the normal range. Importantly, lixisenatide had better gastrointestinal tolerability than liraglutide, and provided the significantly greater reduction in the blood sugar level versus liraglutide [31]. Interestingly, lixisenatide was transported across the BBB at a lower dose than liraglutide, and showed enhanced cAMP at equal dose compared with liraglutide [20]. Therefore, lixisenatide was used for this study. One of the prominent events in the pathogenesis of AD is the formation of abundant deposits of amyloid plaques composed of Ab, which is one of important underlying mechanisms of neurodegeneration in AD. Importantly, there was the significant reduction of plaque load by lixisenatide in 12-month-old APP/PS1/tau female mice. Clearly, the reduction of amyloid plaques in the brain is a very desirable result, which shows that the drug lixisenatide has the effect on plaque clearance, an important factor in AD. Tau in the adult human or animal brain is a phosphoprotein, with an average of about 2 mol of phosphate per mole of protein. While, tau, isolated from the AD brain (usually as neurofibrillary tangles), contains 6e8 mol of phosphate per mole of protein [32] and there is thus little debate about the fact that tau is hyperphosphorylated [33]. Hyperphosphorylated tau is one of important hallmarks of AD and the key component of neurofibrillary tangles [9]. This neurofibrillary pathology suggests a loss of axonal integrity and an eventual decline in connectivity and synapses, a consistent correlate of dementia in AD [34]. Prevention of neurofibrillary tangles has begun to emerge as a viable approach to prevention of neurodegeneration in AD. In our experiment, treatment of lixisenatide reduced neurofibrillary tangles in 12-month-old APP/ PS1/tau female mice. The drug lixisenatide could delay the process of tau hyperphosphorylation. There is evidence from epidemiological studies that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with the significant reduction in the risk for development of AD [35]. The inflammation response becomes neurotoxic due to abnormal production of pro-inflammatory cytokines, which are
enhanced in AD brain [36], such as TNF-a [37], which are induced by activated microglia, can interrupt nerve terminal activity, leading to synapse dysfunction and loss, which correlates with cognitive decline [38]. Furthermore, it has been reported that neuroinflammation accompanies the amyloid and tau pathology of AD; it significantly contributes to the pathogenesis of AD and plays an active role in the neurodegenerative process [39]. In this study, administration of lixisenatide resulted in the significant decrease of inflammation response in 12-month-old APP/PS1/tau female mice, which illustrated that the drug lixisenatide had anti-inflammatory effect in AD. The neurotoxicity of Ab in the brain may be involved in the impairment of protein kinase A (PKA)-cAMP response elementbinding protein (CREB) signaling pathway [40e42]. In this study, lixisenatide could reverse the low levels of phospho-PKA and phospho-CREB in 12-month-old APP/PS1/tau female mice. Therefore, the activation of PKA-CREB signaling pathway might be an important mechanism by which lixisenatide prevents against the amyloid plaques. Activation of p38-Mitogen activated protein kinase (p38-MAPK) in neuronal cells has been associated with response to various stresses, such as inflammation and this kinase is closely related to hyperphosphorylated tau protein in AD [43]. Our experiment showed that lixisenatide could block the high level of phospho-p38-MAPK in 12-month-old APP/PS1/tau female mice, indicating that inhibition of p38-MAPK might be involved in the protective action of lixisenatide against neurofibrillary tangles and neuroinflammation [44]. Lixisenatide, administrated in an APP/PS1/tau mouse model of AD, could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation, through the activation of PKA-CREB signaling pathway and inhibition of p38-MAPK. The study demonstrates that GLP-1R agonists have a range of properties that might be beneficial in treating neurodegenerative conditions. Conflicts of interest There are no conflicts of interest. Acknowledgments Author contributions: Hong-Yan Cai and Jin-Shun Qi designed research; Hong-Yan Cai, Jun-Ting Yang, Zhao-Jun Wang, Mei-Na Wu, Jun Zhang and Wei Yang performed research and data analysis; Hong-Yan Cai, Mei-Na Wu and Jin-Shun Qi wrote the paper. This work was supported by “Sanjin Scholars” of Shanxi Province; The National Natural Science Foundation of China [grant numbers 31471080, 31600865, 31700918]; Higher School Science and Technology Innovation Project of Education Department in Shanxi Province [grant number 2015152]; and The Undergraduate Innovation and Entrepreneurship Project of Shanxi Medical University [grant number 20160109]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.114. References [1] D.J. Selkoe, Alzheimer's disease: genes, proteins, and therapy, Physiol. Rev. 81 (2001) 741e766. [2] E. Gjoneska, A.R. Pfenning, H. Mathys, G. Quon, A. Kundaje, L.H. Tsai, M. Kellis, Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease, Nature 518 (2015) 365e369. [3] W.P. Esler, M.S. Wolfe, A portrait of Alzheimer secretasesenew features and familiar faces, Science 293 (2001) 1449e1454.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7 [4] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics, Science 297 (2002) 353e356. [5] L. Collin, B. Bohrmann, U. Gopfert, K. Oroszlan-Szovik, L. Ozmen, F. Gruninger, Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease, Brain 137 (2014) 2834e2846. [6] W.S. Griffin, J.G. Sheng, M.C. Royston, S.M. Gentleman, J.E. McKenzie, D.I. Graham, G.W. Roberts, R.E. Mrak, Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression, Brain Pathol. 8 (1998) 65e72. [7] R. Brookmeyer, E. Johnson, K. Ziegler-Graham, H.M. Arrighi, Forecasting the global burden of Alzheimer's disease, Alzheimers Dement. 3 (2007) 186e191. [8] M. Schnaider Beeri, U. Goldbourt, J.M. Silverman, S. Noy, J. Schmeidler, R. Ravona-Springer, A. Sverdlick, M. Davidson, Diabetes mellitus in midlife and the risk of dementia three decades later, Neurology 63 (2004) 1902e1907. [9] G. Verdile, S.J. Fuller, R.N. Martins, The role of type 2 diabetes in neurodegeneration, Neurobiol. Dis. 84 (2015) 22e38. [10] S. Mojsov, G.C. Weir, J.F. Habener, Insulinotropin: glucagon-like peptide I (737) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas, J. Clin. Invest. 79 (1987) 616e619. [11] B. Gallwitz, Exenatide in type 2 diabetes: treatment effects in clinical studies and animal study data, Int. J. Clin. Pract. 60 (2006) 1654e1661. [12] A. Vella, P. Shah, A.S. Reed, A.S. Adkins, R. Basu, R.A. Rizza, Lack of effect of exendin-4 and glucagon-like peptide-1-(7,36)-amide on insulin action in nondiabetic humans, Diabetologia 45 (2002) 1410e1415. [13] B. Gallwitz, Therapies for the treatment of type 2 diabetes mellitus based on incretin action, Minerva Endocrinol. 31 (2006) 133e147. [14] T. Perry, H.W. Holloway, A. Weerasuriya, P.R. Mouton, K. Duffy, J.A. Mattison, N.H. Greig, Evidence of GLP-1-mediated neuroprotection in an animal model of pyridoxine-induced peripheral sensory neuropathy, Exp. Neurol. 203 (2007) 293e301. [15] T. Iwai, S. Ito, K. Tanimitsu, S. Udagawa, J. Oka, Glucagon-like peptide-1 inhibits LPS-induced IL-1beta production in cultured rat astrocytes, Neurosci. Res. 55 (2006) 352e360. [16] S. Gengler, P.L. McClean, R. McCurtin, V.A. Gault, C. Holscher, Val(8)GLP-1 rescues synaptic plasticity and reduces dense core plaques in APP/PS1 mice, Neurobiol. Aging 33 (2012) 265e276. [17] M.J. During, L. Cao, D.S. Zuzga, J.S. Francis, H.L. Fitzsimons, X. Jiao, R.J. Bland, M. Klugmann, W.A. Banks, D.J. Drucker, C.N. Haile, Glucagon-like peptide-1 receptor is involved in learning and neuroprotection, Nat. Med. 9 (2003) 1173e1179. [18] T. Vilsboll, H. Agerso, T. Krarup, J.J. Holst, Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects, J. Clin. Endocrinol. Metab. 88 (2003) 220e224. [19] R. Lennox, P.R. Flatt, V.A. Gault, Lixisenatide improves recognition memory and exerts neuroprotective actions in high-fat fed mice, Peptides 61 (2014) 38e47. [20] K. Hunter, C. Holscher, Drugs developed to treat diabetes, liraglutide and lixisenatide, cross the blood brain barrier and enhance neurogenesis, BMC Neurosci. 13 (2012) 33. [21] H.Y. Cai, C. Holscher, X.H. Yue, S.X. Zhang, X.H. Wang, F. Qiao, W. Yang, J.S. Qi, Lixisenatide rescues spatial memory and synaptic plasticity from amyloid beta protein-induced impairments in rats, Neuroscience 277 (2014) 6e13. [22] H.Y. Cai, Z.J. Wang, C. Holscher, L. Yuan, J. Zhang, P. Sun, J. Li, W. Yang, M.N. Wu, J.S. Qi, Lixisenatide attenuates the detrimental effects of amyloid beta protein on spatial working memory and hippocampal neurons in rats, Behav. Brain Res. 318 (2017) 28e35. [23] S. Oddo, A. Caccamo, J.D. Shepherd, M.P. Murphy, T.E. Golde, R. Kayed, R. Metherate, M.P. Mattson, Y. Akbari, F.M. LaFerla, Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction, Neuron 39 (2003) 409e421. [24] S.H. Yang, J. Kim, M.J. Lee, Y. Kim, Abnormalities of plasma cytokines and spleen in senile APP/PS1/Tau transgenic mouse model, Sci. Rep. 5 (2015) 15703. [25] C. Bertoni-Freddari, S.L. Sensi, B. Giorgetti, M. Balietti, G. Di Stefano, L.M. Canzoniero, T. Casoli, P. Fattoretti, Decreased presence of perforated synapses in a triple-transgenic mouse model of Alzheimer's disease,
7
Rejuvenation Res. 11 (2008) 309e313. [26] L.K. Clinton, L.M. Billings, K.N. Green, A. Caccamo, J. Ngo, S. Oddo, J.L. McGaugh, F.M. LaFerla, Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice, Neurobiol. Dis. 28 (2007) 76e82. [27] K.R. Laws, K. Irvine, T.M. Gale, Sex differences in cognitive impairment in Alzheimer's disease, World J. Psychiatry 6 (2016) 54e65. [28] M. Christensen, F.K. Knop, J.J. Holst, T. Vilsboll, Lixisenatide, a novel GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus, IDrugs 12 (2009) 503e513. [29] P.L. McClean, C. Holscher, Lixisenatide, a drug developed to treat type 2 diabetes, shows neuroprotective effects in a mouse model of Alzheimer's disease, Neuropharmacology 86 (2014) 241e258. [30] L. Qi, L. Ke, X. Liu, L. Liao, S. Ke, X. Liu, Y. Wang, X. Lin, Y. Zhou, L. Wu, Z. Chen, L. Liu, Subcutaneous administration of liraglutide ameliorates learning and memory impairment by modulating tau hyperphosphorylation via the glycogen synthase kinase-3beta pathway in an amyloid beta protein induced alzheimer disease mouse model, Eur. J. Pharmacol. 783 (2016) 23e32. [31] C. Kapitza, T. Forst, H.V. Coester, F. Poitiers, P. Ruus, A. Hincelin-Mery, Pharmacodynamic characteristics of lixisenatide once daily versus liraglutide once daily in patients with type 2 diabetes insufficiently controlled on metformin, Diabetes Obes. Metab. 15 (2013) 642e649. [32] H. Ksiezak-Reding, W.K. Liu, S.H. Yen, Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments, Brain Res. 597 (1992) 209e219. [33] M. Goedert, M.G. Spillantini, N.J. Cairns, R.A. Crowther, Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms, Neuron 8 (1992) 159e168. [34] R.D. Terry, E. Masliah, D.P. Salmon, N. Butters, R. DeTeresa, R. Hill, L.A. Hansen, R. Katzman, Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment, Ann. Neurol. 30 (1991) 572e580. [35] P.L. McGeer, M. Schulzer, E.G. McGeer, Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies, Neurology 47 (1996) 425e432. [36] J.K. Ryu, T. Cho, Y.T. Wang, J.G. McLarnon, Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain, J. Neuroinflammation 6 (2009) 39. [37] A.R. Vaz, S.L. Silva, A. Barateiro, A. Fernandes, A.S. Falcao, M.A. Brito, D. Brites, Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin, Exp. Neurol. 229 (2011) 381e390. [38] C. Holmes, C. Cunningham, E. Zotova, D. Culliford, V.H. Perry, Proinflammatory cytokines, sickness behavior, and Alzheimer disease, Neurology 77 (2011) 212e218. [39] P.S. Aisen, K.L. Davis, Inflammatory mechanisms in Alzheimer's disease: implications for therapy, Am. J. Psychiatry 151 (1994) 1105e1113. [40] W.N. Han, C. Holscher, L. Yuan, W. Yang, X.H. Wang, M.N. Wu, J.S. Qi, Liraglutide protects against amyloid-beta protein-induced impairment of spatial learning and memory in rats, Neurobiol. Aging 34 (2013) 576e588. [41] M. Coma, L. Sereno, B. Da Rocha-Souto, T.C. Scotton, J. Espana, M.B. Sanchez, M. Rodriguez, J. Agullo, C. Guardia-Laguarta, M. Garcia-Alloza, L.A. Borrelli, J. Clarimon, A. Lleo, B.J. Bacskai, C.A. Saura, B.T. Hyman, T. Gomez-Isla, Triflusal reduces dense-core plaque load, associated axonal alterations and inflammatory changes, and rescues cognition in a transgenic mouse model of Alzheimer's disease, Neurobiol. Dis. 38 (2010) 482e491. [42] S. Jeon, J.E. Park, J. Lee, Q.F. Liu, H.J. Jeong, S.C. Pak, S. Yi, M.H. Kim, C.W. Kim, J.K. Park, G.W. Kim, B.S. Koo, Illite improves memory impairment and reduces Abeta level in the Tg-APPswe/PS1dE9 mouse model of Alzheimers disease through Akt/CREB and GSK-3beta phosphorylation in the brain, J. Ethnopharmacol. 160 (2015) 69e77. [43] Y. Li, L. Liu, S.W. Barger, W.S. Griffin, Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway, J. Neurosci. 23 (2003) 1605e1611. [44] H. Zhu, Y. Zhang, Z. Shi, D. Lu, T. Li, Y. Ding, Y. Ruan, A. Xu, The neuroprotection of liraglutide against ischaemia-induced apoptosis through the activation of the PI3K/AKT and MAPK pathways, Sci. Rep. 6 (2016) 26859.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease Hong-Yan Cai a, 1, Jun-Ting Yang b, 1, Zhao-Jun Wang b, Jun Zhang b, Wei Yang b, Mei-Na Wu b, *, Jin-Shun Qi b, ** a b
Department of Microbiology and Immunology, Shanxi Medical University, 56 Xinjian South Road, Taiyuan, Shanxi Province, 030001, China Department of Physiology, Shanxi Medical University, 56 Xinjian South Road, Taiyuan, Shanxi Province, 030001, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2017 Accepted 18 November 2017 Available online xxx
Type 2 diabetes mellitus (T2DM) has been identified as a high risk factor for Alzheimer's disease (AD). The impairment of insulin signaling has been found in AD brain. Glucagon-like peptide-1 (GLP-1) is an incretin hormone, normalises insulin signaling and acts as a neuroprotective growth factor. We have previously shown that the long-lasting GLP-1 receptor (GLP-1R) agonist lixisenatide plays an important role in memory formation, synaptic plasticity and cell proliferation of rats. In the follow-up study, we analysed the neuroprotective effect and mechanism of lixisenatide, injected for 60 days at 10 nmol/kg i.p. once daily in APP/PS1/tau female mice and C57BL/6J female mice (as control) aged 12 month. The results showed that lixisenatide could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation in the hippocampi of 12-month-old APP/PS1/tau female mice; activation of PKA-CREB signaling pathway and inhibition of p38-MAPK might be the important mechanisms in the neuroprotective function of lixisenatide. The study demonstrated that GLP-1R agonists such as lixisenatide might have the potential to be developed as a novel therapy for AD. © 2017 Elsevier Inc. All rights reserved.
Keywords: Lixisenatide Alzheimer's disease Amyloid plaques Neurofibrillary tangles Neuroinflammation
1. Introduction Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder, characterized by memory loss and cognitive decline [1]. The pathological features of AD include amyloid plaques, neurofibrillary tangles and neuroinflammation, which are caused by deposits of b-amyloid (Ab) fragments, hyperphosphorylated tau protein and excessive activation of microglia
Abbreviations: T2DM, Type 2 diabetes mellitus; AD, Alzheimer's disease; GLP-1, Glucagon-like peptide-1; GLP-1R, GLP-1 receptor; Ab, b-amyloid; APP, amyloid precursor protein; DPP-4, dipeptidyl peptidase 4; BBB, bloodebrain barrier; APP/ PS1/tau, amyloid precursor protein/presenilin-1/tau protein; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PKA, protein kinase A; CREB, cAMP response element-binding protein; p38-MAPK, p38-Mitogen activated protein kinase; NSAIDs, nonsteroidal anti-inflammatory drugs. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H.-Y. Cai), [email protected] (J.-T. Yang), [email protected] (Z.-J. Wang), [email protected] (J. Zhang), vividmail@ 163.com (W. Yang), [email protected] (M.-N. Wu), [email protected] (J.-S. Qi). 1 With the same contribution.
and astrocyte, respectively [2]. Ab is cleaved from amyloid precursor protein (APP) by b- and g-secretases [3] and is thought to play a central role in the onset and progression of AD, which has led to the amyloid cascade hypothesis [4]. Tau is the axonal protein that normally stabilizes microtubules. Abnormal phosphorylation, misfolding and aggregation of tau lead to neurofibrillary tangle formation and ultimately to neuronal cell death [5]. Activated microglia can lead to neuron damage or death, which can induce further activation of microglia, thus resulting in a self-propagating and detrimental cycle of neuroinflammation and neurodegeneration [6]. Up to now, AD is a huge global burden, because of the lack of effective treatments [7]. Hence, it is imperative to search for new drugs for AD. Epidemiological studies have recently discovered that Type 2 diabetes mellitus (T2DM) is considered as a high-risk factor of AD [8]; T2DM and AD share many clinical and pathological features, one of which is impaired insulin signaling [9]; which opens up the opportunities for developing new treatments of AD. Glucagon-like peptide-1 (GLP-1) is an endogenous 30-amino acid peptide hormone, derived from the processing of proglucagon in intestinal L
https://doi.org/10.1016/j.bbrc.2017.11.114 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
2
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
cells and acts as an incretin to release insulin from pancreatic bcells [10]. It activates a parallel signaling pathway to insulin [11] and does not affect the blood glucose level in normoglycemic people [12,13]. Emerging evidence indicates that GLP-1 acts as a growth factor and has been shown to enhance neurite outgrowth [14]; GLP-1 prevents the release of the inflammatory cytokine IL-1b [15]. Moreover, GLP-1 receptor (GLP-1R) is widely expressed in many regions of CNS, particularly the hippocampus [16]. GLP-1R plays an important role in regulating neuronal plasticity and cell survival. Mice overexpressing GLP-1R in the hippocampus developed increased neurite growth and showed improved learning [17]. However, natural GLP-1 has a half-life of a few minutes as it is broken down by dipeptidyl peptidase 4 (DPP-4) [18]. Thus, it is unsuitable for routine clinical use on account of its short half-life. Lixisenatide, a novel long-lasting GLP-1R agonist, is effective for the treatment of T2DM. It comprises 44 amino acids and is similar in structure to exendin-4 (another GLP-1R agonist), differing by absence of a C-terminal proline at position 38 and addition of six Cterminal lysine residues at position 39. These structural modifications result in the significantly higher affinity for GLP-1R [19]. Importantly, it can cross the bloodebrain barrier (BBB) [20]. Our pervious study showed that lixisenatide rescued spatial learning and memory deficits and hippocampal LTP suppression of rats [21]; lixisenatide effectively inhibited Ab25-35-induced Ca2þ elevation and cytotoxicity in the hippocampal formation cultures [22]. Futhermore, the neuroprotective effect of lixisenatide on AD transgenic mice would be discussed. Therefore, in this study, using immunohistochemical staining and Western blotting technique, we observed whether chronic and systemic administration of lixisenatide could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation in the amyloid precursor protein/presenilin-1/tau protein (APP/PS1/tau) female mouse model of AD at 12 month of age; explored the possible molecular mechanisms. 2. Materials and methods 2.1. Animals Triple amyloid precursor protein/presenilin-1/tau protein (APP/ PS1/tau) AD transgenic mice (MMRRC Stock No: 34830-JAX/3xTgAD), which were obtained from Jackson Laboratory (Bar Harbor, Maine, USA) [23] and then bred in the laboratory animal breeding room, over-expressed mutant APP (APPSWE), PS1 (PS1M146V) and tau (tauP301L). The mutant mouse significantly exhibited amyloid plaques and neurofibrillary tangles between 12 and 15 months [24]. It provided the ideal setting for investigating AD-related neurodegenerative events and showed pathological changes that matched the temporal- and region-specific profiles of what occurred in the brain of AD patients [25]. Because female was related to more serious pathology in AD mice [26,27], APP/PS1/tau female mice from the offspring, were used for the experiments, as well as agematched C57BL/6J (C57) female mice, which were wild-type (WT) as control and were obtained from the experimental animal center in Shanxi Medical University. Mice were maintained on a 12/12 lightedark cycle at temperature 23 ± 1 C in a relative humidity of 60% room. Food and water were available ad libitum. All mice handling and procedures accorded with guidelines of the Shanxi Animal Research Ethics Committee. 2.2. Drug treatment APP/PS1/tau female mice and WT female mice (12-month-old) were randomly assigned to four groups: WT þ saline, APP/PS1/ tau þ saline, WT þ lixisenatide, APP/PS1/tau þ lixisenatide.
Lixisenatide (Sigma, St. Louis, MO, USA), with the purity >99%, analysed by reversed-phase HPLC, was stored in dry form and dissolved in saline (0.9% NaCl) before experiments. In preclinical tests of lixisenatide, the effective dose was 10 nmol/kg bw for lixisenatide [28]. We therefore chose the dose 10 nmol/kg for lixisenatide in order to compare the drug on a similar level. APP/PS1/ tau female mice and WT female mice were injected i.p. with saline or lixisenatide at 10 nmol/kg body weight once daily for 60 days [29]. 2.3. Immunohistochemical staining The mice were sacrificed with heart perfusion fixation with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). Whole-brain tissue was dissected and postfixed within 4% PFA for 24 h. Some of the tissue was dehydrated in 30% sucrose solution and embedded with frozen embedding medium O.C.T. Compound (SAKURA, USA). The brain blocks were sliced into 25-mm-thick coronal sections with the freezing microtome (Leica, CM1850, German). Then, the sections were incubated with 5% hydrogen peroxide at room temperature for 15min and blocked with 5% goat serum (Solarbio, China) for 30min. Brain slices were incubated with the primary antibody overnight at 4 C, and followed by the secondary antibody at 37 C for 2 h. The following primary antibodies were used: mouse monoclonal anti-Ab antibody (dilution 1:500, 803015, Biolegend, USA) and rabbit polyclonal anti-Iba-1 antibody (dilution 1:200, 019-19741, WAKO, Japan). The following secondary antibodies were used: Peroxidase-Conjugated AffiniPure Goat antiRabbit IgG (H þ L) (dilution 1:200, ZB-2301, ZSGB-BIO, China), Peroxidase-Conjugated AffiniPure Goat anti-Mouse IgG (H þ L) (dilution 1:200, ZB-2305, ZSGB-BIO, China). The DAB method was applied for positive area coloration. Some brain tissue was dehydrated in 30% sucrose solution and embedded with paraffin. Brain sections (thickness 2 mm) were prepared with the Leica microtome. Then, the sections were incubated with 3% hydrogen peroxide at room temperature for 15min. Next, they were heated in the micro-oven for 3min with pH 6.0 citrate acid buffer. Sections were washed with PBS (0.5% TritonX100) three times (15min per time) and blocked with 5% goat serum for 30min. Primary antibody, rabbit monoclonal anti-tau (phospho T231) antibody (dilution 1:200, ab151559, Abcam, UK) was incubated at 37 C for 1 h. Secondary antibody, PeroxidaseConjugated AffiniPure Goat anti-Rabbit IgG (H þ L) (dilution 1:200, ZSGB-BIO, China) was incubated at 37 C for 20min. The DAB method was applied for positive cell coloration and positive area coloration. All sections were photographed under the microscope and analysis of percentage stained area and percentage positive cells in the CA1 region of the hippocampus was conducted for amyloid plaques, neurofibrillary tangles and inflammation. The percentage stained area and percentage positive cells of each image (2 images per section, approximately 3-4 sections per mouse, n ¼ 6 mice per group) were quantified with Image-Pro Plus 6.0 (Media Cybernetics, USA). 2.4. Western blotting The hippocampi of mice were dissected. Protein of these tissues was extracted (Tissue Protein Extraction Reagent by Boster, China) and supplemented with complete protease inhibitor (Boster, China). The protein concentration was measured using a bicinchoninic acid protein assay kit after removing debris by low-speed centrifugation. 30 mg sample protein was separated on 12% SDSepolyacrylamide gels. After electrophoresis, protein was transferred onto PVDF membrane and nonspecific binding was
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membrane was incubated with the primary antibody overnight at 4 C, followed by the secondary antibody at room temperature for 2 h. The following primary antibodies were used: rabbit monoclonal anti-phospho-PKA antibody (dilution 1:1000, #5661, Cell Signaling Technology, USA), rabbit monoclonal anti-PKA antibody (dilution 1:1000, #5842, Cell Signaling Technology, USA); rabbit monoclonal anti-phospho-CREB antibody (dilution 1:2500, ab32096, Abcam, UK), rabbit monoclonal antiCREB antibody (dilution 1:1000, ab32515, Abcam, UK); rabbit monoclonal anti-phospho-p38 antibody (dilution 1:1000, #4511, Cell Signaling Technology, USA), rabbit monoclonal anti-p38 antibody (dilution 1:1000, #2387, Cell Signaling Technology, USA). Mouse monoclonal anti-GAPDH antibody (dilution 1:1000, TA-08, ZSGB-BIO, China) was used as loading control. The secondary antibodies were Peroxidase-Conjugated AffiniPure Goat anti-Rabbit IgG (H þ L) (dilution 1:5000, ZB-2301, ZSGB-BIO, China) and Peroxidase-Conjugated AffiniPure Goat anti-Mouse IgG (H þ L) (dilution 1:5000, ZB-2305, ZSGB-BIO, China). The membrane was rinsed with TBST and the immunocomplex was visualized by using an enhanced chemiluminescence detection kit (Beyotime, China). The signals of the membrane were scanned with the FluorChem Scanner and quantified with the Alpha View SA software. 2.5. Statistics All values were displayed as mean ± standard error (SEM). The SPSS 13.0 and SigmaPlot 11.0 statistical packages were used for statistical analyses. The data were examined by a two-way repeated measures analysis of variance (ANOVA). The statistical significance level was set at p < 0.05.
3
3. Results 3.1. Effects of lixisenatide on amyloid plaque load in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on amyloid plaque load in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 7.846, P ¼ 0.041; lixisenatide: F (1,36) ¼ 8.293, P ¼ 0.037; APP/PS1/tau by lixisenatide: F (1,36) ¼ 7.541, P ¼ 0.043). As shown in Fig. 1, Tukey's post hoc tests showed that compared with WT þ saline mice (1.90% ± 0.20%), percentage of Ab-area in the hippocampi of APP/PS1/tau þ saline mice (9.48% ± 1.20%) was increased (P ¼ 0.039); there was no difference in WT þ saline mice and WT þ lixisenatide mice (2.06% ± 0.30%); lixisenatide significantly reduced percentage of Ab-area in the hippocampi of APP/PS1/tau mice (5.87% ± 0.70%), compared to APP/PS1/tau þ saline mice (P ¼ 0.04). 3.2. Effects of lixisenatide on neurofibrillary tangles in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on p-tau positive cell numbers in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 8.984, P ¼ 0.033; lixisenatide: F (1,36) ¼ 8.192, P ¼ 0.038; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.346, P ¼ 0.031). As shown in Fig. 2A and B, Tukey's post hoc tests showed that compared with WT þ saline mice (51.60% ± 4.30%), percentage of p-tau positive cell numbers in the hippocampi of APP/PS1/tau þ saline mice (89.20% ± 6.20%) was
Fig. 1. Lixisenatide reduced amyloid plaques in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of amyloid plaques in four groups. B, Histograms showed that Ab-area of APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; Ab-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
4
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Fig. 2. Lixisenatide reduced neurofibrillary tangles in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of neurofibrillary tangles in four groups. B, Histograms showed that p-tau positive cell numbers of APP/PS1/tau þ saline group were increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; p-tau positive cell numbers of APP/PS1/ tau þ lixisenatide group were decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). C, Histograms showed that p-tau-area of APP/ PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; p-tau-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
increased (P ¼ 0.034); there was no difference in WT þ saline mice and WT þ lixisenatide mice (53.20% ± 3.10%); lixisenatide significantly decreased percentage of p-tau positive cell numbers in the hippocampi of APP/PS1/tau mice (62.30% ± 4.90%), compared to APP/PS1/tau þ saline mice (P ¼ 0.037). Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on p-tau-area in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/ PS1/tau: F (1,36) ¼ 7.431, P ¼ 0.044; lixisenatide: F (1,36) ¼ 6.378, P ¼ 0.049; APP/PS1/tau by lixisenatide: F (1,36) ¼ 8.004, P ¼ 0.039). As shown in Fig. 2 A and C, Tukey's post hoc tests showed that compared with WT þ saline mice (6.56% ± 0.60%), percentage of ptau-area in the hippocampi of APP/PS1/tau þ saline mice (32.58% ± 3.90%) was increased (P ¼ 0.045); there was no difference in WT þ saline mice and WT þ lixisenatide mice (6.62% ± 0.70%); lixisenatide significantly decreased percentage of p-tau-area in the hippocampi of APP/PS1/tau mice (14.99% ± 1.40%), compared to APP/PS1/tau þ saline mice (P ¼ 0.043). Lixisenatide could reduce neurofibrillary tangles in the hippocampi of APP/PS1/tau female mice.
3.3. Effects of lixisenatide on neuroinflammation in the hippocampi of 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on activation of microglia in the hippocampi of 12-month-old APP/PS1/tau female mice (APP/PS1/tau: F (1,36) ¼ 9.981, P ¼ 0.027; lixisenatide: F (1,36) ¼ 9.491, P ¼ 0.029; APP/PS1/tau by lixisenatide: F (1,36) ¼ 7.920, P ¼ 0.039). As shown in Fig. 3, Tukey's post hoc tests
showed that compared with WT þ saline mice (3.67% ± 0.60%), percentage of Iba-1-area in the hippocampi of APP/PS1/tau þ saline mice (16.85% ± 1.90%) was increased (P ¼ 0.033); there was no difference in WT þ saline mice and WT þ lixisenatide mice (4.17% ± 0.60%); lixisenatide significantly decreased percentage of Iba-1-area in the hippocampi of APP/PS1/tau mice (7.41% ± 0.90%), compared to APP/PS1/tau þ saline mice (P ¼ 0.037). Lixisenatide could reduce neuroinflammation in the hippocampi of APP/PS1/tau female mice.
3.4. Lixisenatide relieved the suppression of PKA-CREB signaling pathway in 12-month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pPKA (APP/PS1/tau: F (1,36) ¼ 6.984, P ¼ 0.046; lixisenatide: F (1,36) ¼ 8.492, P ¼ 0.035; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.398, P ¼ 0.03). As shown in Fig. 4 A and D, Tukey's post hoc tests showed that compared with WT þ saline mice (71.34% ± 7.90%), the level of p-PKA in the hippocampi of APP/PS1/ tau þ saline mice (28.52% ± 3.50%) was decreased (P ¼ 0.042); there was no difference in WT þ saline mice and WT þ lixisenatide mice (69.16% ± 7.10%); lixisenatide significantly reversed the low level of p-PKA in the hippocampi of APP/PS1/tau mice (57.07% ± 6.30%), compared to APP/PS1/tau þ saline mice (P ¼ 0.039). Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pCREB (APP/PS1/tau: F (1,36) ¼ 7.164, P ¼ 0.045; lixisenatide: F (1,36) ¼ 9.458, P ¼ 0.03; APP/PS1/tau by lixisenatide: F (1,36) ¼ 9.344, P ¼ 0.031). As shown in Fig. 4 B and E, Tukey's post
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
5
Fig. 3. Lixisenatide decreased activation of microglia in the hippocampi of APP/PS1/tau female mice. A, Representative immunohistochemical images of activation of microglia in four groups. B, Histograms showed that Iba-1-area of APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; Iba-1-area of APP/PS1/tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group, 3-4 sections per mouse). *P < 0.05.
Fig. 4. Lixisenatide reversed the suppression of PKA-CREB signaling pathway and inhibited the activation of p38-MAPK in APP/PS1/tau female mice. A, B and C, The expression of p-PKA, PKA, p-CREB, CREB, p-p38 and p38 in four groups. D and E, Histograms showed that levels of p-PKA and p-CREB in APP/PS1/tau þ saline group were decreased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; levels of p-PKA and p-CREB in APP/ PS1/tau þ lixisenatide group were increased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group). F, Histograms showed that the level of p-p38 in APP/PS1/tau þ saline group was increased, compared to WT þ saline group; there was no significant difference between WT þ saline group and WT þ lixisenatide group; the level of p-p38 in APP/PS1/ tau þ lixisenatide group was decreased, compared to APP/PS1/tau þ saline group (n ¼ 6 mice per group). *P < 0.05.
hoc tests showed that compared with WT þ saline mice (59.30% ± 6.80%), the level of p-CREB in the hippocampi of APP/PS1/
tau þ saline mice (22.53% ± 4.50%) was decreased (P ¼ 0.036); there was no difference in WT þ saline mice and WT þ lixisenatide mice
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
6
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
(58.70% ± 7.10%); lixisenatide significantly relieved the decrease level of p-CREB in the hippocampi of APP/PS1/tau mice (42.70% ± 3.50%), compared to APP/PS1/tau þ saline mice (P ¼ 0.042). 3.5. Lixisenatide inhibited the activation of p38-MAPK in 12month-old APP/PS1/tau female mice Two-way ANOVA showed APP/PS1/tau female mice and lixisenatide treatment had significant main effects on the level of pp38 (APP/PS1/tau: F (1,36) ¼ 7.904, P ¼ 0.04; lixisenatide: F (1,36) ¼ 7.452, P ¼ 0.044; APP/PS1/tau by lixisenatide: F (1,36) ¼ 8.358, P ¼ 0.036). As shown in Fig. 4C and F, Tukey's post hoc tests showed that compared with WT þ saline mice (11.30% ± 2.50%), the level of p-p38 in the hippocampi of APP/PS1/ tau þ saline mice (53.11% ± 3.90%) was increased (P ¼ 0.043); there was no difference in WT þ saline mice and WT þ lixisenatide mice (12.30% ± 2.70%); lixisenatide significantly inhibited the increase level of p-p38 in the hippocampi of APP/PS1/tau mice (36.20% ± 3.30%), compared to APP/PS1/tau þ saline mice (P ¼ 0.038). 4. Discussion GLP-1R agonists could be taken by non-diabetic people as they only had the effect on the blood sugar level in hyperglycaemia [20]. The study showed that body weight, plasma glucose, plasma insulin and hippocampal insulin in the AD mouse model, were not affected by liraglutide (another long-lasting GLP-1R agonist) treatment [30]. So, lixisenatide also did not promote insulin secretion when the plasma glucose level was in the normal range. Importantly, lixisenatide had better gastrointestinal tolerability than liraglutide, and provided the significantly greater reduction in the blood sugar level versus liraglutide [31]. Interestingly, lixisenatide was transported across the BBB at a lower dose than liraglutide, and showed enhanced cAMP at equal dose compared with liraglutide [20]. Therefore, lixisenatide was used for this study. One of the prominent events in the pathogenesis of AD is the formation of abundant deposits of amyloid plaques composed of Ab, which is one of important underlying mechanisms of neurodegeneration in AD. Importantly, there was the significant reduction of plaque load by lixisenatide in 12-month-old APP/PS1/tau female mice. Clearly, the reduction of amyloid plaques in the brain is a very desirable result, which shows that the drug lixisenatide has the effect on plaque clearance, an important factor in AD. Tau in the adult human or animal brain is a phosphoprotein, with an average of about 2 mol of phosphate per mole of protein. While, tau, isolated from the AD brain (usually as neurofibrillary tangles), contains 6e8 mol of phosphate per mole of protein [32] and there is thus little debate about the fact that tau is hyperphosphorylated [33]. Hyperphosphorylated tau is one of important hallmarks of AD and the key component of neurofibrillary tangles [9]. This neurofibrillary pathology suggests a loss of axonal integrity and an eventual decline in connectivity and synapses, a consistent correlate of dementia in AD [34]. Prevention of neurofibrillary tangles has begun to emerge as a viable approach to prevention of neurodegeneration in AD. In our experiment, treatment of lixisenatide reduced neurofibrillary tangles in 12-month-old APP/ PS1/tau female mice. The drug lixisenatide could delay the process of tau hyperphosphorylation. There is evidence from epidemiological studies that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with the significant reduction in the risk for development of AD [35]. The inflammation response becomes neurotoxic due to abnormal production of pro-inflammatory cytokines, which are
enhanced in AD brain [36], such as TNF-a [37], which are induced by activated microglia, can interrupt nerve terminal activity, leading to synapse dysfunction and loss, which correlates with cognitive decline [38]. Furthermore, it has been reported that neuroinflammation accompanies the amyloid and tau pathology of AD; it significantly contributes to the pathogenesis of AD and plays an active role in the neurodegenerative process [39]. In this study, administration of lixisenatide resulted in the significant decrease of inflammation response in 12-month-old APP/PS1/tau female mice, which illustrated that the drug lixisenatide had anti-inflammatory effect in AD. The neurotoxicity of Ab in the brain may be involved in the impairment of protein kinase A (PKA)-cAMP response elementbinding protein (CREB) signaling pathway [40e42]. In this study, lixisenatide could reverse the low levels of phospho-PKA and phospho-CREB in 12-month-old APP/PS1/tau female mice. Therefore, the activation of PKA-CREB signaling pathway might be an important mechanism by which lixisenatide prevents against the amyloid plaques. Activation of p38-Mitogen activated protein kinase (p38-MAPK) in neuronal cells has been associated with response to various stresses, such as inflammation and this kinase is closely related to hyperphosphorylated tau protein in AD [43]. Our experiment showed that lixisenatide could block the high level of phospho-p38-MAPK in 12-month-old APP/PS1/tau female mice, indicating that inhibition of p38-MAPK might be involved in the protective action of lixisenatide against neurofibrillary tangles and neuroinflammation [44]. Lixisenatide, administrated in an APP/PS1/tau mouse model of AD, could reduce amyloid plaques, neurofibrillary tangles and neuroinflammation, through the activation of PKA-CREB signaling pathway and inhibition of p38-MAPK. The study demonstrates that GLP-1R agonists have a range of properties that might be beneficial in treating neurodegenerative conditions. Conflicts of interest There are no conflicts of interest. Acknowledgments Author contributions: Hong-Yan Cai and Jin-Shun Qi designed research; Hong-Yan Cai, Jun-Ting Yang, Zhao-Jun Wang, Mei-Na Wu, Jun Zhang and Wei Yang performed research and data analysis; Hong-Yan Cai, Mei-Na Wu and Jin-Shun Qi wrote the paper. This work was supported by “Sanjin Scholars” of Shanxi Province; The National Natural Science Foundation of China [grant numbers 31471080, 31600865, 31700918]; Higher School Science and Technology Innovation Project of Education Department in Shanxi Province [grant number 2015152]; and The Undergraduate Innovation and Entrepreneurship Project of Shanxi Medical University [grant number 20160109]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.11.114. References [1] D.J. Selkoe, Alzheimer's disease: genes, proteins, and therapy, Physiol. Rev. 81 (2001) 741e766. [2] E. Gjoneska, A.R. Pfenning, H. Mathys, G. Quon, A. Kundaje, L.H. Tsai, M. Kellis, Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease, Nature 518 (2015) 365e369. [3] W.P. Esler, M.S. Wolfe, A portrait of Alzheimer secretasesenew features and familiar faces, Science 293 (2001) 1449e1454.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114
H.-Y. Cai et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7 [4] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics, Science 297 (2002) 353e356. [5] L. Collin, B. Bohrmann, U. Gopfert, K. Oroszlan-Szovik, L. Ozmen, F. Gruninger, Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease, Brain 137 (2014) 2834e2846. [6] W.S. Griffin, J.G. Sheng, M.C. Royston, S.M. Gentleman, J.E. McKenzie, D.I. Graham, G.W. Roberts, R.E. Mrak, Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression, Brain Pathol. 8 (1998) 65e72. [7] R. Brookmeyer, E. Johnson, K. Ziegler-Graham, H.M. Arrighi, Forecasting the global burden of Alzheimer's disease, Alzheimers Dement. 3 (2007) 186e191. [8] M. Schnaider Beeri, U. Goldbourt, J.M. Silverman, S. Noy, J. Schmeidler, R. Ravona-Springer, A. Sverdlick, M. Davidson, Diabetes mellitus in midlife and the risk of dementia three decades later, Neurology 63 (2004) 1902e1907. [9] G. Verdile, S.J. Fuller, R.N. Martins, The role of type 2 diabetes in neurodegeneration, Neurobiol. Dis. 84 (2015) 22e38. [10] S. Mojsov, G.C. Weir, J.F. Habener, Insulinotropin: glucagon-like peptide I (737) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas, J. Clin. Invest. 79 (1987) 616e619. [11] B. Gallwitz, Exenatide in type 2 diabetes: treatment effects in clinical studies and animal study data, Int. J. Clin. Pract. 60 (2006) 1654e1661. [12] A. Vella, P. Shah, A.S. Reed, A.S. Adkins, R. Basu, R.A. Rizza, Lack of effect of exendin-4 and glucagon-like peptide-1-(7,36)-amide on insulin action in nondiabetic humans, Diabetologia 45 (2002) 1410e1415. [13] B. Gallwitz, Therapies for the treatment of type 2 diabetes mellitus based on incretin action, Minerva Endocrinol. 31 (2006) 133e147. [14] T. Perry, H.W. Holloway, A. Weerasuriya, P.R. Mouton, K. Duffy, J.A. Mattison, N.H. Greig, Evidence of GLP-1-mediated neuroprotection in an animal model of pyridoxine-induced peripheral sensory neuropathy, Exp. Neurol. 203 (2007) 293e301. [15] T. Iwai, S. Ito, K. Tanimitsu, S. Udagawa, J. Oka, Glucagon-like peptide-1 inhibits LPS-induced IL-1beta production in cultured rat astrocytes, Neurosci. Res. 55 (2006) 352e360. [16] S. Gengler, P.L. McClean, R. McCurtin, V.A. Gault, C. Holscher, Val(8)GLP-1 rescues synaptic plasticity and reduces dense core plaques in APP/PS1 mice, Neurobiol. Aging 33 (2012) 265e276. [17] M.J. During, L. Cao, D.S. Zuzga, J.S. Francis, H.L. Fitzsimons, X. Jiao, R.J. Bland, M. Klugmann, W.A. Banks, D.J. Drucker, C.N. Haile, Glucagon-like peptide-1 receptor is involved in learning and neuroprotection, Nat. Med. 9 (2003) 1173e1179. [18] T. Vilsboll, H. Agerso, T. Krarup, J.J. Holst, Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects, J. Clin. Endocrinol. Metab. 88 (2003) 220e224. [19] R. Lennox, P.R. Flatt, V.A. Gault, Lixisenatide improves recognition memory and exerts neuroprotective actions in high-fat fed mice, Peptides 61 (2014) 38e47. [20] K. Hunter, C. Holscher, Drugs developed to treat diabetes, liraglutide and lixisenatide, cross the blood brain barrier and enhance neurogenesis, BMC Neurosci. 13 (2012) 33. [21] H.Y. Cai, C. Holscher, X.H. Yue, S.X. Zhang, X.H. Wang, F. Qiao, W. Yang, J.S. Qi, Lixisenatide rescues spatial memory and synaptic plasticity from amyloid beta protein-induced impairments in rats, Neuroscience 277 (2014) 6e13. [22] H.Y. Cai, Z.J. Wang, C. Holscher, L. Yuan, J. Zhang, P. Sun, J. Li, W. Yang, M.N. Wu, J.S. Qi, Lixisenatide attenuates the detrimental effects of amyloid beta protein on spatial working memory and hippocampal neurons in rats, Behav. Brain Res. 318 (2017) 28e35. [23] S. Oddo, A. Caccamo, J.D. Shepherd, M.P. Murphy, T.E. Golde, R. Kayed, R. Metherate, M.P. Mattson, Y. Akbari, F.M. LaFerla, Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction, Neuron 39 (2003) 409e421. [24] S.H. Yang, J. Kim, M.J. Lee, Y. Kim, Abnormalities of plasma cytokines and spleen in senile APP/PS1/Tau transgenic mouse model, Sci. Rep. 5 (2015) 15703. [25] C. Bertoni-Freddari, S.L. Sensi, B. Giorgetti, M. Balietti, G. Di Stefano, L.M. Canzoniero, T. Casoli, P. Fattoretti, Decreased presence of perforated synapses in a triple-transgenic mouse model of Alzheimer's disease,
7
Rejuvenation Res. 11 (2008) 309e313. [26] L.K. Clinton, L.M. Billings, K.N. Green, A. Caccamo, J. Ngo, S. Oddo, J.L. McGaugh, F.M. LaFerla, Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice, Neurobiol. Dis. 28 (2007) 76e82. [27] K.R. Laws, K. Irvine, T.M. Gale, Sex differences in cognitive impairment in Alzheimer's disease, World J. Psychiatry 6 (2016) 54e65. [28] M. Christensen, F.K. Knop, J.J. Holst, T. Vilsboll, Lixisenatide, a novel GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus, IDrugs 12 (2009) 503e513. [29] P.L. McClean, C. Holscher, Lixisenatide, a drug developed to treat type 2 diabetes, shows neuroprotective effects in a mouse model of Alzheimer's disease, Neuropharmacology 86 (2014) 241e258. [30] L. Qi, L. Ke, X. Liu, L. Liao, S. Ke, X. Liu, Y. Wang, X. Lin, Y. Zhou, L. Wu, Z. Chen, L. Liu, Subcutaneous administration of liraglutide ameliorates learning and memory impairment by modulating tau hyperphosphorylation via the glycogen synthase kinase-3beta pathway in an amyloid beta protein induced alzheimer disease mouse model, Eur. J. Pharmacol. 783 (2016) 23e32. [31] C. Kapitza, T. Forst, H.V. Coester, F. Poitiers, P. Ruus, A. Hincelin-Mery, Pharmacodynamic characteristics of lixisenatide once daily versus liraglutide once daily in patients with type 2 diabetes insufficiently controlled on metformin, Diabetes Obes. Metab. 15 (2013) 642e649. [32] H. Ksiezak-Reding, W.K. Liu, S.H. Yen, Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments, Brain Res. 597 (1992) 209e219. [33] M. Goedert, M.G. Spillantini, N.J. Cairns, R.A. Crowther, Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms, Neuron 8 (1992) 159e168. [34] R.D. Terry, E. Masliah, D.P. Salmon, N. Butters, R. DeTeresa, R. Hill, L.A. Hansen, R. Katzman, Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment, Ann. Neurol. 30 (1991) 572e580. [35] P.L. McGeer, M. Schulzer, E.G. McGeer, Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies, Neurology 47 (1996) 425e432. [36] J.K. Ryu, T. Cho, Y.T. Wang, J.G. McLarnon, Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain, J. Neuroinflammation 6 (2009) 39. [37] A.R. Vaz, S.L. Silva, A. Barateiro, A. Fernandes, A.S. Falcao, M.A. Brito, D. Brites, Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin, Exp. Neurol. 229 (2011) 381e390. [38] C. Holmes, C. Cunningham, E. Zotova, D. Culliford, V.H. Perry, Proinflammatory cytokines, sickness behavior, and Alzheimer disease, Neurology 77 (2011) 212e218. [39] P.S. Aisen, K.L. Davis, Inflammatory mechanisms in Alzheimer's disease: implications for therapy, Am. J. Psychiatry 151 (1994) 1105e1113. [40] W.N. Han, C. Holscher, L. Yuan, W. Yang, X.H. Wang, M.N. Wu, J.S. Qi, Liraglutide protects against amyloid-beta protein-induced impairment of spatial learning and memory in rats, Neurobiol. Aging 34 (2013) 576e588. [41] M. Coma, L. Sereno, B. Da Rocha-Souto, T.C. Scotton, J. Espana, M.B. Sanchez, M. Rodriguez, J. Agullo, C. Guardia-Laguarta, M. Garcia-Alloza, L.A. Borrelli, J. Clarimon, A. Lleo, B.J. Bacskai, C.A. Saura, B.T. Hyman, T. Gomez-Isla, Triflusal reduces dense-core plaque load, associated axonal alterations and inflammatory changes, and rescues cognition in a transgenic mouse model of Alzheimer's disease, Neurobiol. Dis. 38 (2010) 482e491. [42] S. Jeon, J.E. Park, J. Lee, Q.F. Liu, H.J. Jeong, S.C. Pak, S. Yi, M.H. Kim, C.W. Kim, J.K. Park, G.W. Kim, B.S. Koo, Illite improves memory impairment and reduces Abeta level in the Tg-APPswe/PS1dE9 mouse model of Alzheimers disease through Akt/CREB and GSK-3beta phosphorylation in the brain, J. Ethnopharmacol. 160 (2015) 69e77. [43] Y. Li, L. Liu, S.W. Barger, W.S. Griffin, Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway, J. Neurosci. 23 (2003) 1605e1611. [44] H. Zhu, Y. Zhang, Z. Shi, D. Lu, T. Li, Y. Ding, Y. Ruan, A. Xu, The neuroprotection of liraglutide against ischaemia-induced apoptosis through the activation of the PI3K/AKT and MAPK pathways, Sci. Rep. 6 (2016) 26859.
Please cite this article in press as: H.-Y. Cai, et al., Lixisenatide reduces amyloid plaques, neurofibrillary tangles and neuroinflammation in an APP/PS1/tau mouse model of Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/ j.bbrc.2017.11.114