Involvement of insulin signalling pathway in methamphetamine-induced hyperphosphorylation of Tau

Accepted Manuscript Title: Involvement of insulin signalling pathway in methamphetamine-induced hyperphosphorylation of Tau Authors: Huaisha Xu, Xiaodong Chen, Jun Wang, Tingyu Yang, Na Liu, Jie Cheng, Rong Gao, Jingli Liu, Hang Xiao PII: DOI: Reference:

S0300-483X(18)30133-1 https://doi.org/10.1016/j.tox.2018.07.002 TOX 52055

To appear in:

Toxicology

Received date: Revised date: Accepted date:

29-3-2018 31-5-2018 3-7-2018

Please cite this article as: Xu H, Chen X, Wang J, Yang T, Liu N, Cheng J, Gao R, Liu J, Xiao H, Involvement of insulin signalling pathway in methamphetamine-induced hyperphosphorylation of Tau, Toxicology (2018), https://doi.org/10.1016/j.tox.2018.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Involvement of insulin signalling pathway in methamphetamine-induced hyperphosphorylation of Tau

Huaisha Xua,1, Xiaodong Chenb,1, Jun Wangc, Tingyu Yangc, Na Liud, Jie Chengc, Rong Gaoe,

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Jingli Liuf, Hang Xiaoc, *

Division of Psychiatry, Medical School, Nanjing University, Nanjing, 210093, China

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Department of Anesthesiology, the First Affiliated Hospital of Nanjing Medical University,

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Nanjing, 210029, China

Department of Toxicology, the Key Lab of Modern Toxicology (NJMU), Ministry of

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Education, School of Public Health, Nanjing Medical University, Nanjing, 211166, China

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University, Nanjing, 210029, China

Department of Hygienic Analysis and Detection, the Key Lab of Modern Toxicology

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e

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Department of Medical Psychology, Nanjing Brain Hospital, Nanjing Medical

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(NJMU), Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, 211166, China

Department of Experimental Medicine, Drum Tower Hospital, Nanjing University Medical

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f

School, Nanjing 210008, China

Corresponding author at: Hang Xiao, Nanjing Medical University, 818 Tianyuan East Road,

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*

Nanjing 211166, China. Tel.: +86 25 86868431; fax: +86 25 86868499

E-mail address: [email protected] (H. Xiao)

Highlights

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METH treatment disturbs the insulin signaling in the neural cells.

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METH also causes the hyperphosphorylation of AD-related pathological protein p-tau.

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METH-induced tau phosphorylation mediated via the insulin signalling pathway and downstream key kinase GSK3β.

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These authors have contributed equally to this study.

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Abstract

Methamphetamine (METH), an amphetamine-like drug, is one of the most commonly used

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central nervous system psychostimulants worldwide. METH abuse frequently leads to

cognitive decline and dementia-like changes, but the mechanisms remain poorly understood. In the present study, the mechanisms of METH-induced changes in Alzheimer's disease-like

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pathological protein in Neuro2A cells were explored. Our results indicated that METH

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exposure significantly increased the expression of the pathological protein

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hyperphosphorylated tau (p-tau). Further analysis revealed that METH exposure obviously disrupted insulin signalling, resulted in brain insulin resistance, which manifested as

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downregulation of the insulin receptor substrate-1, AKTser 473, and GSK3β activation. Notably,

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the linkage between p-tau expression and insulin signalling can be partially verified by treatment with the insulin-sensitizing drug rosiglitazone and GSK3β inhibitor TWS119 which

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specifically reversed METH-induced hyperphosphorylation of tau.

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Abbreviations

METH, methamphetamine; N2a, Neuro2A; IRS1, insulin receptor substrate-1; AKT, protein kinase B; GSK3β, glycogen synthase kinase-3β; AD, Alzheimer’s disease; MEM, minimum essential medium; 5-HT, serotonin; PD, Parkinson's disease; Aβ, amyloid-beta; BBB, blood-brain

barrier; IR, insulin receptor; PI3K, phosphatidylinositol-3-kinase; APP, amyloid precursor protein; HD, Huntington's disease

Our results indicate that insulin signalling can be therapeutically exploited for attenuating

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METH-induced upregulation of p-tau. Keywords: Methamphetamine; Insulin signalling pathway; Alzheimer’s disease; Tau;

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neurodegenerative damage

1. Introduction

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The World Drug Report 2017 suggested that users of amphetamines have reached 37 million

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globally. Among amphetamines, methamphetamine (METH) represents the greatest health

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threat. A high dosage of METH may frequently lead to serious consequences, including renal

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and liver failure, cardiac arrhythmias, heart attacks, cerebrovascular haemorrhages, and stroke

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(Davidson et al., 2001; Sato, 1992; Srisurapanont et al., 2003). As chronic abusive uses of METH have increased in recent years, METH-induced neurodegenerative changes, such as

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cognitive decline and dementia-like changes, have attracted the attention of researchers (Hart

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et al., 2012; Potvin et al., 2018).

Abnormal hyperphosphorylation of tau protein is found in a wide variety of dementias,

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including Alzheimer’s disease, frontotemporal dementia, progressive supranuclear palsy, and corticobasilar degeneration (Ballatore et al., 2007). Therefore, hyperphosphorylation of tau is a crucial pathological protein in neurodegeneration (Yoshiyama et al., 2013). Some principal kinases play pivotal roles in hyperphosphorylation of tau, such as glycogen synthase kinase3β (GSK-3β) (Ferrer et al., 2005). Our previous findings indicated that the insulin signalling

cascade induced by some neurotoxic environmental endocrine disruptors activated GSK3β phosphorylation, contributing to p-tau enhancement and causing Alzheimer’s disease (AD)like neurotoxicity (Wang et al., 2017). Studies showed that insulin signalling pathway function is essential for neuronal survival within brains by maintaining appropriate energy

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metabolism homeostasis, and thus affects learning and memory abilities (Plum et al., 2005; Reagan, 2007). Therefore, disruption of brain insulin signalling such as brain insulin

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resistance may contribute to neurodegenerative-like diseases (de la Monte, 2017). Based on previous studies by our laboratory and others, we hypothesized that the insulin signalling

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pathway is involved in METH-induced tau hyperphosphorylation. In the present study, we

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found that METH exposure significantly disrupted insulin signalling, which may contribute to

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upregulation of p-tau. These finds reveal previously unrecognized METH-induced brain

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neurodegenerative-like disease.

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damage, providing a potential strategy for therapeutic intervention in METH-induced

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2. Materials and methods

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2.1. Materials

Methamphetamine (National Institute for Food and Drug Control, Beijing, China) was dissolved in double-distilled water as a stock solution and then diluted in minimum essential

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medium (MEM) to a final concentration of 100 mM. The dosage of methamphetamine was determined by the cell counting kit-8 assay. The GSK3β inhibitor TWS119 (Selleck, Houston, TX, USA) was dissolved in dimethyl sulfoxide as a stock solution and then diluted in MEM immediately before use at a concentration of 1000 nM. Rosiglitazone (Sigma, St Louis, MO,

USA) was dissolved in dimethyl sulfoxide as a stock solution and then diluted in MEM to a final concentration of 50 μM.

2.2. Cell culture

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Mouse neuroblastoma Neuro2A (N2a) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). N2a cells were cultured in MEM supplemented

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with 10% foetal bovine serum, 80 U/mL penicillin, and 80 μg/mL streptomycin at 37℃ in a humidified atmosphere of 5% CO2.

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2.3. Cell counting kit-8 assay

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Cell counting kit-8 was purchased from Dojindo (Kumamoto, Japan). N2a cells were seeded

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in 96-well culture plates at a density of 1 × 104 cells per well and treated with varied

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concentrations of METH. After incubation for 12 h at 37°C, and cell proliferation was

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assessed by absorbance detection at 450 nm with a NanoDrop 1000 spectrophotometer

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(Thermo Fisher Scientific, Waltham, MA, USA).

2.4. Western blot analysis

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Total proteins in N2a cells were extracted in RIPA buffer (Sigma) containing protease inhibitor and phosphatase inhibitor cocktails (1:500, Sigma). Protein concentrations were

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evaluated by the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Proteins were separated by electrophoresis on an 8% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and blocked with 5% non-fat milk or 1% bovine serum albumin for 1 h at room temperature

(eg 25°C). The membrane was incubated overnight at 4℃ with primary antibody in Trisbuffered saline containing 0.1% Tween and 5% non-fat milk or 1% bovine serum albumin.

The following primary antibodies were used: antibodies against pSer307-IRS1 (1:500, Cell Signaling Technology, Danvers, MA, USA), IRS1 (1:1000, Cell Signaling Technology),

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pSer473-AKT (1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling

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Technology), pTyr216-GSK3β (1:1000, Cell Signaling Technology), GSK3β (1:1000, Cell

Signaling Technology), pSer199-tau (1:5000, Abcam, Cambridge, UK), pSer214-tau (1:2000, Abcam), pSer396-tau (1:20 000, Abcam), GAPDH (1: 5000, Affinity, USA). The membranes

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were washed and incubated at room temperature with anti-mouse or antirabbit horseradish

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peroxidase conjugated to IgG (1:30 000, Jackson ImmunoResearch Laboratories, West Grove,

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PA, USA). The results were normalized to those of GAPDH and analysed using ImageJ

2.5. Statistical analysis

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software (NIH, Bethesda, MD, USA).

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Data are expressed as the means ± SEM for all experiments. The statistical significance of the

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differences was determined by one-way analysis of variance and Dunnett multiple comparison procedures using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA). All tests of

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statistical significance were two-sided and P < 0.05 was considered significant.

3. Results

3.1. Involvement of insulin pathway in methamphetamine-induced hyperphosphorylation of tau

Brain insulin resistance has been suggested to be closely associated to neurodegenerative disease. To examine whether the insulin signalling cascade participates in METH-induced Tau phosphorylation, N2a cells were exposed to METH (0, 100, 300, and 900 μM) for 12 h, and insulin pathway protein expression was examined by western blot analysis. We chose a

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dose range based on the clinical drug concentration of METH chronic abusers and used CCK8 validation (Abdul Muneer et al., 2011; Carson et al., 2012; Fisher et al., 2015; Parikh et al.,

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2015). As depicted in Fig. 1A, METH dose-dependently upregulated the phosphorylation of insulin receptor substrate-1 (IRS1) (Ser307), a phosphorylation site that inhibits tyrosine

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phosphorylation, thereby decreasing insulin signalling transduction (Hancer et al., 2014).

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Additionally, another key insulin signalling protein, kinase B (AKT) (Ser473), was examined.

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Our results indicated that exposure of N2a cells to METH decreased the expression of AKT

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(Ser473), with a peak response at a concentration of 900 μM. Methamphetamine's inhibition

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of the insulin signalling pathway was dose-dependently enhanced when the dose administered was similar to that detected in the blood of the drug abuser. To assess the time course of

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METH action, the cells were incubated with METH for 0, 6, 12, and 24 h, and then the levels

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of IRS-1 (Ser307) and AKT (Ser473) were detected. As shown in Fig. 1B, METH exposure significantly increased the expression of IRS-1 (Ser307) at 6 and 12 h, after which protein expression decreased to a steady level. METH treatment markedly decreased the expression

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of AKT (Ser473), with the peak effect observed at 12 h.

Hyperphosphorylation of tau causes neuronal dysfunction, such as microtubule severing, axonal transport impairment, and synapse loss, leading to types of dementia such as AD (Ittner and Gotz, 2011; Takashima, 2008). Numerous studies have shown that GSK3β plays a

pivotal role in modulating tau phosphorylation (Gomez de Barreda et al., 2010; Sun et al., 2002). In our results, pY216-GSK3β phosphorylation was substantially increased, with the peak effect observed at 12 h (Fig. 2A). After establishing that METH exposure contributed to pY216-GSK3β phosphorylation, we next examined the downstream pathological protein p-

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tau. To validate the specific effects of METH on p-tau expression, several phosphorylated sites of tau protein were detected, as shown in Fig. 2A. METH exposure obviously increased

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the expression of pS199-tau, pS214-tau, and pS396-tau, with the maximum increase

occurring at 12 h (P < 0.05). These results indicate that METH significantly inhibited insulin

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signalling and induced downstream GSK3β and tau hyperphosphorylation.

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3.2. Inhibition of phosphorylation of GSK3β reduces the production of METH-induced

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hyperphosphorylation of tau

To gain insight into the association between GSK3β and tau phosphorylation, we examined

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whether TWS119, an inhibitor of GSK3β, could mitigate the METH-dependent

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hyperphosphorylation of tau in N2a cells. As depicted in Fig. 3, treatment with TWS119 dose-dependently downregulated the phosphorylation of GSK3β（Tyr216）at 12 h. Thus, the

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cells were treated with or without GSK3β inhibitor TWS119 (1000 nM) for 2 h and then stimulated with or without METH for 12 h. Pretreatment of the cells with TWS119

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significantly attenuated the METH-induced hyperphosphorylation of tau, including at the Ser199, Ser214, and Ser396 phosphorylation sites. These results indicate that inhibition of phosphorylation of GSK3β (Tyr216, at least in part, attenuates METH-induced hyperphosphorylation of tau.

3.3. Effects of insulin-sensitizing drug rosiglitazone on methamphetamine-induced pathological protein expression Rosiglitazone is a peroxisome proliferator-activated receptor-γ agonist that sensitizes the insulin signal pathway and reduces insulin resistance (Yang et al., 2005). To further examine

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the specific effects of the insulin signal pathway on METH-induced hyperphosphorylation of

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tau, rosiglitazone was used to determine the specificity of central insulin resistance induced by METH and downstream tau pathology induced by central insulin resistance. N2a cells were treated with or without METH (900 μM) and rosiglitazone (50 μM) for 12 h.

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The western blot results showed that METH markedly increased the expression of pS307-

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IRS1 and pY216-GSK3β and decreased the expression of pS473-AKT compared to those in

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the control cultures. Insulin resistance was significantly improved by treatment with rosiglitazone; the enhancement of pS307-IRS1 and pY216-GSK3β expression and

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downregulation of pS473-AKT mediated by METH was significantly ameliorated by

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rosiglitazone (Fig. 4A), indicating that rosiglitazone rescued METH-mediated insulin pathway perturbation. Because insulin resistance in the brain is closely associated with varied

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dementias, we next examined the impact of rosiglitazone on tau phosphorylation. N2a cells were co-incubated with rosiglitazone and METH. As shown in Fig. 4B, METH-induced tau

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hyperphosphorylation including at Ser199, Ser214, and Ser396 was substantially decreased, suggesting that insulin signalling plays important roles in METH-mediated hyperphosphorylation of tau, while rosiglitazone decreased p-tau expression through sensitization of insulin signalling.

4. Discussion

Several studies have shown that chronic abuse of methamphetamine causes severe degenerative damage to the central nervous system(Brooks et al., 2016). A meta-analysis based on 17 cross-sectional studies revealed that the cognitive abilities of methamphetamine abusers were significantly

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reduced(Scott et al., 2007). More recently, a study showed that METH exposure significantly enhanced

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amyloid-beta (Aβ) levels in brain microvascular endothelial cells (Liu et al., 2017), validating that

METH exposure contributes to AD-like pathological changes. However, whether other pathological protein and mechanisms involving METH-induced AD-like undergo pathological changes remains

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largely unclear.

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Insulin receptors in the brain are found at high densities in the CA1 hippocampal field and

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dentate gyrus. These regions are particularly important for learning and memory (De Felice and Benedict, 2015; De Felice, 2013; Gerozissis, 2008). Recently, the linkage between insulin

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signalling and neurodegenerative diseases in the brain have gained attention. IRS1 is one of

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the most critical nodes in the upstream insulin signalling pathway and the phosphorylation site Ser307 regulates insulin resistance (Copps et al., 2010; O’Neill, 2013). In AD patients,

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IRS-1 and the downstream AKT protein are significantly decreased in the brain (Talbot et al., 2012). Other investigators reported that using selective aging manipulation to delay

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neurodegenerative diseases such as AD and Huntington's diseases (HD) (El-Ami et al., 2014), targeting the elimination of the serine loci in the phosphorylation of IRS1 can protect nematodes from AD and HD-related protein induced toxicity (de Barreda et al., 2010). In agreement with these observations, our data showed a significant increase in IRS-1 Ser307 (inhibition of the insulin signalling transduction) protein expression and pronounced

inhibition of the downstream signalling by AKT Ser 473 after METH treatment. As an integration site of multiple neural pathways, abnormal expression of PI3K/AKT in the neurons is one of the early features of AD (O’Neill, 2013). Selective downregulation of AKT Ser phosphorylation has been observed in the lymphoblast cells of familial AD(Ryder et al.,

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2004). Moreover, immunohistochemistry analysis revealed that AKT, GSK3β, and p-tau colocalize in some neurofibrillary tangles (Yarchoan et al., 2014). These results, taken together,

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supported our hypothesis that METH exposure contributes to disturbance of the insulin signalling pathway, which may result in AD-like changes.

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Notably, GSK3β is an important downstream molecule in the insulin signalling pathway

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and a key mediator of tau phosphorylation. Overexpression of GSK3β in mice showed serious

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dentate gyrus atrophy, microtubule dysfunction, and tau hyperphosphorylation (Yarchoan et al., 2014; Zhang et al., 2013). This suggests that GSK3β specifically activates multiple sites

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on the tau protein, including Ser199, Ser214, Ser396, Thr205, and Thr231 (Wang et al.,

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2012). In the present study, we examined several phosphorylation sites on p-tau after treatment with METH, including Ser199, Ser214, and Ser396, which further confirmed the

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crucial effects of GSK3β in METH-induced upregulation of the AD associated protein. Additionally, following treatment with the GSK3β blocker TWS119, METH-induced

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upregulation of p-tau expression was markedly attenuated.

The tau protein, a natively unfolded microtubule-associated protein, regulates its binding affinity for microtubules and stabilizes microtubules by phosphorylating several critical serine and threonine residues (Grundke-Iqbal et al., 1986; Kadavath et al., 2015). Intriguingly, increased levels of p-tau facilitate the degradation of microtubule-associated protein,

contributing to neural damage. Neurofibrillary tangles composed of hyperphosphorylated tau are important characteristics of early AD (Ballatore et al., 2007; Simic et al., 2016). Notably, tau phosphorylation at Ser396 was shown to be one of the earliest events in AD (MondragonRodriguez et al., 2014); in our results, METH exposure obviously enhanced the p-tauSer396

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level, suggesting the adverse effects of METH exposure on neurons. A series of studies reported that targeting of pathological tau in AD has high potential in animal models (Collin

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et al., 2014; Gu et al., 2013; Panza et al., 2016; Pedersen and Sigurdsson, 2015; Schroeder et

al., 2016; Zhang et al., 2015). These findings suggest that tau is a therapeutic target for AD. In

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the current study, we found that the insulin signalling pathway may play important roles in p-

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tau regulation, as intervention in the insulin signalling pathway by rosiglitazone, a

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thiazolidinedione agent developed to improve insulin resistance by enhancing insulin

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sensitivity, directly attenuated the abnormal phosphorylation of tau protein and reversed the

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toxic effects of METH (Escribano et al., 2010). In accordance with our study, a recent study demonstrated the importance of the protective effects of insulin signalling pathways in

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METH-induced neural damage, showing that insulin, which enhances insulin signalling,

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effectively relieved METH-induced anxiety-like behaviour and neuroinflammation in animals (Beirami et al., 2017). These studies suggest that the insulin signalling pathway should be

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further examined to decrease METH-induced neural damage.

5. Conclusion

The present study outlined the molecular mechanism of METH-mediated neural damage involving the disturbance of insulin signalling and hyperphosphorylation of tau, which may lead to the formation of neurofibrillary tangles and ultimately culminate into AD-like disease

(Fig. 5). However, the vitro results with Alzheimer Disease-like effects is speculative. Still to be explored is that pathophysiological implications of p-tau changes. Collectively, these findings reveal a previously unrecognized pathway by which METH-induced AD-like disease is regulated by insulin signalling.

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Conflicts of interest statement The authors declare that there are no conflicts of interest.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81673213,

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81202230, 81500613), Natural Science Foundation of Jiangsu Province (BK20151557,

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BK20171500, BK20150080, BK20150108), Program for Key disease of Jiangsu Province

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Science and Technology Department (BL2014088), Program for Innovative Medical

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Research Team of Jiangsu Province (CXTDA2017007), Key Disciplines of Jiangsu Province

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(ZDXKC2016006), and General Project of Nanjing Medical Science and Technology

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Figure legends

Figure 1. Treatment with METH disturbed the insulin signaling pathway in N2a cells.

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Western blot expression analysis of (A) different concentrations of METH (0, 100, 300 and 900 μM) on insulin receptor substrate-1 (IRS1) phosphorylation and protein kinase B (AKT) phosphorylation for 12h. (B) IRS1 and AKT phosphorylation at various time points (0, 6, 12

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and 24h) of 900 μM METH treatment. GAPDH was used as loading control. Mean values ± SEMs are representative of three independent samples. Significant differences between the treatment groups and the control group were determined via one-way ANOVA and the

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Dunnett multiple comparison procedure. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Figure 2. Treatment with METH enhanced the expression of phosphorylated glycogen synthase kinase-3β (GSK3β) and phosphorylated tau in N2a cells. Western blot analysis indicated the expression of phosphorylated GSK3β and phosphorylated tau at various time

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points (0, 6, 12 and 24h) of 900 μM METH treatment. GAPDH was used as loading control. Mean values ± SEMs are representative of three independent samples. Significant differences between the treatment groups and the control group were determined via oneway ANOVA and the Dunnett multiple comparison procedure. *p < 0.05, **p < 0.01 vs control.

Figure 3. Inhibition of glycogen synthase kinase-3β (GSK3β) reduces the expression of tau phosphorylation in N2a cells. (A) Effect of TWS119 on the expression of GSK3β

phosphorylation. (B) Effect of TWS119 on the expression of GSK3β and tau phosphorylation induced by METH. GAPDH was used as loading control. Mean values ± SEMs are representative of three independent samples. Significant differences between the treatment groups and the control group were determined via one-way ANOVA and the Dunnett multiple comparison procedure. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, ###p < 0.001 vs control. Figure 4. Effects of rosiglitazone on METH-induced insulin signaling pathway and tau phosphorylation in N2a cells. (A) Effect of rosiglitazone on the expression of insulin

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receptor substrate-1 (IRS1) phosphorylation and protein kinase B (AKT) phosphorylation medicated by METH. (B) Effect of rosiglitazone on the expression of glycogen synthase kinase-3β (GSK3β) and tau phosphorylation medicated by METH. GAPDH was

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used as loading control. Mean values ± SEMs are representative of three independent

samples. Significant differences between the treatment groups and the control group were determined via one-way ANOVA and the Dunnett multiple comparison procedure. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, ###p < 0.001 vs control.

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Figure 5. Hypothetical model of METH-induced Alzheimer’s disease like neurotoxicology. IR, insulin receptor; IRS-1, insulin receptor substrate-1; AKT, protein kinase B; GSK3β,

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glycogen synthase kinase-3β; NFTs, neurofibrillary tangles; PPARγ, peroxisome

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proliferator-activated receptor-γ

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Supplementary Figure 1. Using cell counting kit-8 to validate the dosage of methamphetamine. N2a cells were incubated with varied concentrations of METH for 24 h. The absorbance was measured by using a microplate reader at a wavelength of 450 nm.

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Mean values ± SEMs are representative of three independent samples. *p < 0.05, **p < 0.01,

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***p < 0.001 vs control.

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