Diabetes mellitus and Alzheimer’s disease: GSK-3β as a potential link

Accepted Manuscript Title: Diabetes mellitus and Alzheimer’s disease: GSK-3␤ as a potential link Authors: Ying Zhang, Nan-qu Huang, Fei Yan, Hai Jin, Shao-yu Zhou, Jing-shan Shi, Feng Jin PII: DOI: Reference:

S0166-4328(17)31529-2 https://doi.org/10.1016/j.bbr.2017.11.015 BBR 11174

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

13-9-2017 8-10-2017 13-11-2017

Please cite this article as: Zhang Ying, Huang Nan-qu, Yan Fei, Jin Hai, Zhou Shaoyu, Shi Jing-shan, Jin Feng.Diabetes mellitus and Alzheimer’s disease: GSK-3␤ as a potential link.Behavioural Brain Research https://doi.org/10.1016/j.bbr.2017.11.015 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.

Diabetes mellitus and Alzheimer's disease: GSK-3β as a potential link

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Ying Zhang1†, Nan-qu Huang2†, Fei Yan1, Hai Jin3 , Shao-yu Zhou1,4, Jing-shan Shi1, Feng Jin1* 1. Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Guizhou, China 2. Drug Clinical Trial Institution, The First People’s Hospital of Zunyi, Guizhou, China 3. Institute of Digestive Diseases of Affiliated Hospital, Zunyi Medical University, Guizhou, China 4. Department of Environmental Health, Indiana University Bloomington, Indiana, United States. * Corresponding author. †

These authors have contributed equally to this work.

Highlights

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E-mail address: [email protected].

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• GSK-3β is involved in the physiological and pathological progress of DM and AD respectively. • In DM, GSK-3β is one of the key factors leading to insulin deficiency and insulin resistance. • In AD, GSK-3β plays an important role in hyperphosphorylation of microtubuleassociated protein tau. • GSK-3β as a potential link between DM and AD is reviewed. • GSK-3β as a link between DM and AD further supports that AD is regarded as Type 3 diabetes. Abstract: It is well known that Alzheimer’s disease (AD) is closely related to diabetes mellitus (DM), and AD is also regarded as Type 3 diabetes (T3D). However, the exact link between AD and DM is still unclear. Recently, more and more evidence has shown

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that glycogen synthase kinase-3β (GSK-3β) may be the potential link between DM and AD. In DM, GSK-3β is the crucial enzyme of glycogen synthesis, which plays a key

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role in regulating blood glucose. More importantly, GSK-3β is one of the key factors leading to insulin deficiency and insulin resistance, and insulin resistance is an

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important hallmark of the occurrence and development of DM. In AD, GSK-3β plays an important role in hyperphosphorylation of microtubule-associated protein tau (tau), which is one of the pathological features in AD. GSK-3β is one of the important kinases of tau phosphorylation and is involved in the insulin/phosphoinositide 3-kinase/protein kinase B (insulin/PI3K/Akt) signaling pathway. Dysfunction of the insulin/PI3K/Akt signaling pathway, which regulates glucose metabolism in the brain, can lead to tau hyperphosphorylation in the brain of AD patents. Additionally, insulin resistance in DM

may cause β-amyloid (Aβ) deposition, which will be cleared by tau, but excessive phosphorylation of tau will further aggravate the neurotoxicity; then damage the brain and affect the cognitive function. GSK-3β is considered as a common kinase in insulin signaling transduction and tau protein phosphorylation, so we have reasons to believe that GSK-3β is a potential link between DM and AD.

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Abbreviations: 3xTg, triple-transgenic; Aβ, β-amyloid; AD, Alzheimer’s disease; ADAS-cog, Alzheimer's Disease Assessment Scale-cognitive subscale; Akt, protein kinase B; APP, amyloid precursor protein; CREB, cAMP-response element binding protein; DM, diabetes mellitus; FFAs, free fatty acids; GCA, Global Clinical Assessment; GDS, Geriatric Depression Scale; GPCRs, G protein-coupled receptors; GS, glycogen synthase; GSK-3β, glycogen synthase kinase-3β; IGF-1, insulin-like growth factor-1; iNOS, inducible nitric oxide synthase; IRS-1, insulin receptor substrate-1; JNK, C-Jun-N terminal kinase; MMSE, Mini-Mental Status Examination; NFTs, neurofibrillary tangles; NOD, non-obese diabetic; PDK1, phosphoinositidedependent protein kinase 1; PDX-1, duodenal homeobox 1; PI3K, phosphoinositide 3kinase; PP-1, protein phosphatase 1; PS1, presenilin 1; SPs, senile plaques; STAT3, signal transduction and transcriptional activator 3; T3D, Type 3 diabetes; tau, microtubule-associated protein tau; UCP2, uncoupling protein 2.

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Key words: glycogen synthase kinase-3β; diabetes mellitus; Alzheimer’s disease; microtubule-associated protein tau; insulin resistance Introduction

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1.

AD is a neurodegenerative disease which is pathologically characterized by typically senile plaques (SPs) formed of Aβ deposition, and neurofibrillary tangles (NFTs) composed of tau hyperphosphorylation [1, 2]. With increasingly aging

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population, the incidence of AD has increased year by year, therefore, prevention and control of AD has become globally focused [3]. Although a lot of research has been

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carried out on AD, the etiology and pathogenesis of AD are not yet fully understood. In our previous study about icariin (ICA, extracted from Chinese herb Berberidaceae

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epimedium L.) protecting amyloid precursor protein/presenilin 1 (APP/PS1) doubletransgenic AD mice, we also found that 14-month-old AD model mice had significantly higher blood glucose levels than that of wild-type mice of the same age [4]. This suggests that glucose metabolism disorder is associated with AD. In addition, Vandal M, et al. found that there was obvious glucose intolerance in the triple-transgenic mouse model of AD (3xTg-AD) [5]. But, a randomized, double-blind, placebo-controlled trial showed that in patients with early AD, it is very common that they are not diagnosed

with pre-diabetes and diabetes [6]. Interestingly, AD-related proteins will in turn play a role in the signaling pathway of insulin. Many researchers found that the APL-1, an APP-related protein, is encoded by Caenorhabditis elegans gene which also affects the signaling pathway of insulin in AD patients [7]. In addition, researchers from Tohoku University in Japan found that, the way insulin signals work in the brain may be similar to the way insulin signals work in the pancreas of DM patients, suggesting that AD may be described as a diabetic disorder in the brain [8]. Therefore, AD is considered to be

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brain-specific T3D, which is similar to but different from DM [9, 10]. Although the

specific mechanism is still unclear, recent studies have found that GSK-3β associated

2.

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with insulin signaling pathway may be a potential link between AD and DM. Role of GSK-3β in DM

DM is a group of metabolic diseases characterized by high blood sugar, and hyperglycemia is caused by insulin secretion deficiency or its biological effect’s

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damage. GSK-3β as one of the first kinases capable of phosphorylating glycogen

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synthase (GS), is isolated from rabbit skeletal muscle and is one of the few protein

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kinases that are inactivated by phosphorylation [11]. Among them, the high expression of GSK-3β was correlated with the decrease of insulin sensitivity, and it was involved

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in blood glucose regulation, insulin deficiency and insulin resistance. Therefore, more and more attention has been paid to the study of the relationship between GSK-3β and

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the occurrence of DM.

2.1 GSK-3β in blood glucose regulation Blood glucose regulation is not only an important part of the regulation of life’s

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activities but also an important condition for homeostasis in the body. The disorder of blood glucose regulation increased the incidence of DM. Insulin-based regulation is still the main path of blood glucose regulation, in which the insulin receptor-mediated

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signaling pathway plays a key role [12]. PI3K/Akt/GSK-3β signaling pathway is involved in the insulin signaling transduction, and GSK-3β is regulated and controlled

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by insulin in this signaling pathway, which is related to the glycogen synthesis regulation. The insulin receptor binds insulin and has a tyrosine-protein kinase activity, and mediates the metabolic functions of insulin. Binding to insulin stimulates the association of the receptor with downstream mediators, including insulin receptor substrate-1 (IRS-1) and PI3K, the insulin receptor can activate PI3K either directly by binding to the p85 regulatory subunit, which produces PIP3, directly or indirectly leading to phosphorylation and activation of Akt, afterwards Akt phosphorylates the

Ser9 site of GSK-3β and inhibits its activity [13, 14]. Then failing to inhibit the activity of glycogen synthase, which promotes glycogen synthesis and reduces the blood glucose level. When insulin and insulin-like growth factor-1 (IGF-1) signal dysfunction, the body increases GSK-3β activity to eventually increase blood glucose, by phosphorylation of Tyr216 site of GSK-3β to inhibit the activity of PI3K/Akt [15], and also by blockade of Wnt signaling pathway to negatively regulate GSK-3β through PI3K/Akt dependent mechanism [16] (Figure 1). Studies have shown that heat shock-

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induced glycogen synthesis in L6 skeletal muscle cells leads to an increase in glycogen-

associated protein phosphatase 1 (PP-1) and GS activity, accompanied by sustained

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Akt/GSK-3β phosphorylation. When Wortmannin (PI3K inhibitor) inhibits Akt/GSK3β phosphorylation, it prevents 2-deoxyglucose uptake, and eliminates heat shockinduced glycogen synthesis as well [17]. In the DM model mice, it has been found that

excessive activation of GSK-3β results in the decrease of islet β cell proliferation [18].

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Islet β cells are endocrine cells that secrete insulin in the body, which can regulate blood

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glucose levels. Endogenous GSK-3β controls the growth of islet β cells by feedback

blood glucose.

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2.2 GSK-3β and insulin deficiency

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inhibition of the PI3K/Akt signaling pathway, then plays a critical role in regulating

Congenital or acquired dysfunction of islet β cells can lead to inadequate insulin

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secretion, causing DM. The exact mechanism is unclear, but it may be related to glucose toxicity, lipid toxicity, inflammatory response, oxidative stress and other factors [1921]. GSK-3β is one of the key factors that mediate islet β cells apoptosis and is closely

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related to insulin deficiency. A previous study found that hyperglycemia-induced loss of islet β cells is associated with increased oxidative stress [22]. Gliclazide is a sulfonylurea oral anti-diabetic drug preventing human islet β cells from apoptosis

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induced by high glucose, which was due, at least in part, to its antioxidant activity [23]. Under diabetic conditions, the increase in oxidative stress leads to the activation

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of the C-Jun-N terminal kinase (JNK) pathway, further stimulating the phosphorylation of GSK-3β [24, 25], while GSK-3β hyperactivity has anti-proliferative and proapoptotic properties effects on the body. GSK-3β phosphorylation can inhibit the activity of cAMP-response element binding protein (CREB), and then down-regulate the expression of Bcl-2 [26, 27], activation of caspase-3 and caspase-9, leading to reduction of islet β cells, eventually speeding up the process of DM. In addition, inducible nitric oxide synthase (iNOS) as the main medium of inflammation, can

activate JNK and GSK-3β, and inhibit the expression of pancreatic and duodenal homeobox 1 (PDX-1) and IRS-2 protein, finally leading to islet β cell exhaustion [28, 29]. Increased blood glucose levels induce mitochondrial superoxide production and activation of uncoupling protein 2 (UCP2), and reduce the ATP/ADP ratio, leading to impaired β cell function and reduced insulin secretion [30]. Among them, GSK-3β, as one of the rate-limiting enzymes, is a key factor in ATP production that inhibits glycogen synthesis. In addition, glucose tolerance and islet β cells were proliferated

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relative to the control group in β-Gsk-3β-/- mice [31]. Therefore, excessive activation of GSK-3β is associated with apoptosis of islet β cells and reduction of insulin

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secretion, which may lead to DM (Figure 1). 2.3 GSK-3β and insulin resistance

Insulin resistance refers to the decrease of glucose uptake and utilization efficiency drops for various reasons. In order to maintain the stability of blood sugar in the body,

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islet β cells compensate with excessive secretion of insulin and this eventually leads to

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hyperinsulinemia. It’s easy to lead to metabolic syndrome and T2D. Cells regulate

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blood glucose through insulin receptors binding to insulin, and insulin resistance means that the body's cells (mainly muscle) are less sensitive to insulin. Impaired signal

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transduction of the insulin/PI3K/Akt signaling pathway is one of the main features of insulin resistance, and insulin resistance will further lead to an increase in GSK-3β [32,

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33]. Yokoo et al. found the increased phosphorylation of GSK-3β-Ser9 by suppression of constitutively active GSK-3 in cultured bovine adrenal chromaffin cells, and the stability of insulin receptor mRNA was reduced, which in turn affected the level of insulin receptor [34]. In the study of the role of STAT3 (signal transduction and

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transcriptional activator 3) in insulin signaling, L-STAT-/- mice had an increased GSK3β level compared with the control group. It was also found that suppression of GSK-

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3β with lithium chloride and L803-mts can restore glucose balance and improve glucose intolerance [35]. In addition, evidence has shown that insulin resistance is associated

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with hepatitis C virus (HCV) infection, and GSK-3β is also involved in it [36]. HCV E2 protein inhibits glucose uptake and glycogen synthesis by affecting the expression of IRS-1, Akt protein and GSK-3β phosphorylation in Huh7 cells, leading to insulin resistance [37]. It's interesting that inhibition of GSK-3β activity in DM mice can prevent DM-related pathological changes [38]. What’s more, there is another view. Some researchers have suggested that the root cause of T2D metabolism disorder is the disorder of fat metabolism. A large amount of

fat is deposited in muscle, liver and islet β cells, leading to insulin resistance and islet cell dysfunction. Adipose tissue-derived cytokines can cause systemic insulin resistance, and GSK-3β plays an important role in it [39, 40]. In 1994, Benjamin WB et al found that the GSK-3β content in the cytoplasm was reduced during the differentiation of 3T3-L1 cells into adipocytes [41]. It's interesting that the constructed GSK-3β-specific RNAi adenovirus vector was transfected into HEK293A cells, and the expression of GSK-3β was down-regulated, which reduced the effect of free fatty acids

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(FFAs) on human umbilical vein endothelial cells injury [42]. GSK-3β-specific RNAi and lithium chloride also inhibits the activity of GSK-3β in human L02 and HepG2

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cells, and inhibits the upregulation of JNK phosphorylation and Bax uptake by saturated FFAs sodium palmitate and reduces lipoapoptosis [43]. This indicates that GSK-3β is one of the important factors involved in lipid metabolism.

Studies have shown that plasma levels of FFAs are elevated in most obese subjects,

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whereas physiological elevation of plasma FFAs leads to insulin-induced glucose

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transport and/or phosphorylation deficits, which in turn inhibits glucose uptake [44].

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Leonard H Storlien et al found that insulin sensitivity in rat skeletal muscle was negatively correlated with triglyceride levels in muscle, and high-fat diets could lead to

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insulin resistance [45]. Subsequent studies have shown that changes in plasma FFAs concentrations mediate insulin resistance, plasma insulin levels, and glucose

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intolerance [46]. This suggests that obesity and T2D are associated with insulin resistance. In obese individuals, adipose tissue can release a large number of FFAs, glycerol, hormones, proinflammatory cytokines and others involved in the development

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of insulin resistance [47]. Compared with the control group, the expression of GSK-3β protein in the adipose tissue of the insulin-resistant rats induced by high-fat diet was significantly increased [48]. In DM mice, intraperitoneal injection of melatonin can

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improve glucose tolerance and insulin resistance, and increase liver glycogen synthesis, which is related to the level of GSK-3β phosphorylation [49]. HepG2 cells were treated

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with palmitate to induce insulin resistance, and NO-1886 (ibrolipim, a lipoprotein lipase activator) reduced GSK-3β protein levels and stimulated glycogen synthesis, improved insulin resistance [50]. Glucose and FFAs together promote the islet β cell’s apoptosis by activating GSK-3β. When GSK-3β expression is reduced, islet β cell apoptosis decreases and instead its proliferation increases, as does glycogen synthesis, and insulin resistance and fat metabolic disorders. In summary, GSK-3β plays an important role in insulin resistance caused by exogenous infection and endogenous lipid

metabolism disorders [36, 37, 49, 50] (Figure 2). 3.

GSK-3β, tau phosphorylation and AD The function of GSK-3 was originally thought to be the phosphorylation of only

GS and therefore inactivated GS. However, later, a large number of studies have shown that GSK-3 can also phosphorylate tau protein [51, 52]. Two subtypes of GSK-3 were found in mammals: GSK-3α and GSK-3β, the latter of which plays a key role in tau protein phosphorylation. Excessive activation of GSK-3β promotes abnormal

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hyperphosphorylation of tau protein, aggravates degeneration of neurons, interferes with normal synaptic plasticity, and accelerates AD pathology process in AD patients

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[53]. GSK-3β plays an important role in the pathological changes of tau protein in AD

[54]. The activation of GSK-3β was demonstrated to promote abnormal hyperphosphorylation of tau and was involved in the occurrence and development of AD [55]. Microtubules composed of microtubule-associated proteins and tubulin are

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important components of the cytoskeleton, and axonal cytoskeleton is closely related

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to axonal growth. Spittaels K et al found that co-expression of GSK-3β decreased

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axonal dilatation and reduced axonal lesions in the CNS of tau transgenic mice [56]. A decrease in the activity of this kinase inhibits the increase in motile mitochondria, which

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in turn regulates mitochondrial axonal transport [57]. Therefore, phosphorylation of tau by GSK-3β may be critical for axonal elongation during development, also GSK-3β

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phosphorylates the tau protein at multiple sites in intact cells. Furthermore, GSK-3βmediated phosphorylation of Ser404 protects the drosophila visual system [58], and studies have shown that phosphorylation-incompetent tau protein is more serious for

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synaptic damage than hyperphosphorylated tau protein, and surprisingly found that phosphorylation can prolong the longevity of drosophila [59]. In a study published in the Science, Lars Ittner et al proposed a new view that the reason for tau

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phosphorylation is actually to protect neurons from damage. In the early stages of the disease, the protein assists in the protective phosphorylation of tau and interferes with

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the neurotoxic complex formed by Aβ; and they suggest that tau protein phosphorylation initially has a protective effect on neurons, but Aβ can attack the protective function until the function is gradually lost. At this stage, the level of toxicity will lead to neuronal destruction and cognitive impairment associated with AD [60]. Tau phosphorylation plays an important role in the brain, which may be an important mechanism of repair and resistance to injury, but tau hyperphosphorylation is also an important factor in the formation of NFTs. It is quite necessary to control the

phosphorylation of tau in a reasonable range by regulating GSK-3β signaling pathway in AD. 3.1 GSK-3β, an important kinase for phosphorylation of tau protein In 1975, the tau protein was first identified as a thermostable protein necessary for microtubule assembly, which has been shown to have a characterization of native unfolding proteins [61-63]. The tau protein interacts with tubulin to stabilize the microtubules and promote tubulin assembly into microtubules. This study found that

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the tau protein binds to the surface of the microtubule and acts as a velocity "controller" to regulate the transport of dynein and kinesin. This transport is necessary to transfer

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other proteins from the neuronal synapses to the cell body and maintain healthy neurons

[64]. It has been reported that there are about 30 Ser and Thr phosphorylation sites in normal tau proteins [65], and it is worth mentioning that the normal phosphorylation of tau protein is a necessary condition for its normal biological function. As early as 1997,

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Lesort M et al found that neuronal cells rich in phosphorylated tau protein were more

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tolerant to apoptosis [66]. Tau protein phosphorylation is regulated by many kinases

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and phosphatases, and GSK-3β is one of the most important phosphorylated kinases catalyzing tau protein, which plays a key role in regulating tau phosphorylation levels

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and stabilizing microtubules [67]. GSK-3β is highly expressed in the brain tissues, especially in neurons, and its expression in developing brain tissue is much higher than

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that in adult brain tissue [68, 69]. Moreover, the enzyme plays an important role in the growth of axons during brain development [70]. In addition, tau and GSK-3β coimmunoprecipitation were found by chromatographic separation, indicating that the

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kinase binds to tau in the brain, which in turn leads to GSK-3β through tau associated with microtubules [71]. 3.2 GSK-3β, mutant PS1 and tau hyperphosphorylation

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PS1 is an important component of γ-secretase, which is highly expressed in the

hippocampus, inner olfactory cortex, cortex and cerebellum. PS1 protein mutation is

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thought to be closely related to the pathogenesis of AD [72]. Mutant PS1 is easily cleaved by caspases, which increases Aβ content in neurons and then regulates phosphorylation of microtubule-associated protein tau [73]. Previously, Murayama M et al found that overexpression of PS1 reduced the level of cytoplasmic β-catenin in transfected cells, but the consequences of this interaction were unknown [74]. Subsequently, studies have found that wild-type PS1-induced overexpression increases the binding of β-catenin to GSK-3β and accelerates the turnover of endogenous β-

catenin, suggesting that GSK-3β-dependent degradation of β-catenin requires PS1 expression. In contrast, PS1 mutants interfere with this process, which not only attenuate the interaction between GSK-3β and β-catenin, but also interfere with the conversion of β-catenin [75]. Studies have shown that tau and GSK-3β bind to the same region of PS1, and PS1 mutations increase the binding of tau to GSK-3β and its taudirected kinase activity, while the activation of GSK-3β further leads to sustained enhancement of tau hyperphosphorylation and NFTs formation [76]. Further studies

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showed that there were Congo red birefringence and Thioflavin T reactivity in the

neurons of familial Alzheimer’s disease (FAD) mutant (I213T) PS1 knock-in mouse

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which are both histologic criteria of NFTs, indicating that PS1 mutations accelerate the formation of tau pathology and lead to the pathogenesis of AD [77]. GSK-3β-mediated phosphorylation induced PS1 structure changes, reducing the interaction of PS1 with β-catenin, thereby causing the β-catenin to stabilize [78], also it has been demonstrated

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that PS1 is a substrate of inactive GSK-3β [79]. When the mouse overexpresses GSK-

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3β, the hippocampal neurons show a decrease in nuclear β-catenin levels, tau

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hyperphosphorylation, and further neurodegeneration [80]. In summary, the mutant PS1 may induce GSK-3β overexpression, resulting in tau hyperphosphorylation (Figure

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3).

3.3 PI3K/Akt/GSK-3β signaling pathway in tau hyperphosphorylation disorders

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PI3K/Akt/GSK-3β

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hyperphosphorylation. PI3K has the activity of Ser/Thr kinase as well as that of phosphatidylinositol kinase; when PI3K is activated by signals from tyrosine kinases

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and G protein-coupled receptors (GPCRs), a second messenger PIP3 is generated on the cell membrane; PIP3 binds to the N-terminal PH domain of Akt and promotes phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylated Akt protein on

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the Thr308 and Ser473, thereby activating Akt. GSK-3β is a downstream molecule of the PI3K/Akt signaling pathway. Activated Akt binds to GSK-3β, induces GSK-3β to

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transpose into cell membrane, phosphorylates its N-terminal Ser9 active site and inactivates GSK-3β, which in turn affects the downstream substrate of GSK-3β such as tau protein and insulin receptor. Studies have shown that the PI3K/Akt/GSK-3β signaling pathway plays a key role in neuroprotection and enhances cell survival by stimulating cell proliferation and inhibiting apoptosis [81, 82]. However, dysfunction of this signaling pathway will increase GSK-3β activity and lead to excessive phosphorylation of tau. The expression of GSK-3β and the phosphorylation of the tau

protein were detected by transfection of GSK-3β into rat brain. It was found that the abnormal phosphorylation of tau protein was co-located with GSK-3β, indicating that GSK-3β-induced hyperphosphorylation of tau protein was associated with the mechanism of neurodegenerative disease [83]. When the rats were injected with propofol, Akt decreased, GSK-3β phosphorylation increased, and hippocampal apoptosis and cognitive deficits were detected. Pretreatment of dexmedetomidine (Dex) in rats can restore PI3K/Akt/GSK-3β signaling to normal and reduce hippocampal cell

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apoptosis and improve cognitive dysfunction [84]. It has been shown that arctigenin (Arctigenin, a lignan from Wikstroemia indica) has neuroprotective effects on AD mice,

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which can improve learning and memory deficiencies by increasing the

phosphorylation of PI3K/Akt, decreasing the activity of GSK-3β, and decreasing levels of tau phosphorylation of Thr181, Thr231 and Ser404 sites in the hippocampus [85]. The study further confirms that the PI3K/Akt/GSK-3β signaling pathway is the key to

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regulating the phosphorylation of tau protein. In both in vivo, and in vitro, after

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HEK293/tau cells were treated with H2O2, the level of tau phosphorylation in Thr231

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and Ser396 sites increased, but the level of tau phosphorylation decreased to normal after HEK293/tau cells were treated with TPPU, a drug that has been shown to have

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neuroprotective effects. Additionally, TPPU up-regulated the expression of p-Akt, while decreasing the phosphorylation of GSK-3β [86]. Moreover, the neuroprotective

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effect of ginkgolides (Ginkgolides are biologically active terpenic lactones present in Ginkgo biloba) is also related to this pathway. Ginkgolide activates the PI3K/Akt signaling pathway to inhibit the phosphorylation of GSK3β at the Ser9 site, thereby

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protecting cells from tau hyperphosphorylation related toxicity [87]. Thus, both in vivo and in vitro the tau hyperphosphorylation is associated with the PI3K/Akt/GSK-3β signaling pathway, and inhibition of the GSK-3β over-activation may alleviate tau

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hyperphosphorylation, therefore slowing the further aggravation of AD. 4.

GSK-3β, a potential link between DM and AD

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We have known that GSK-3β plays a key role in DM, being the key to the dynamic

regulation of blood glucose, and also being one of the key factors leading to insulin deficiency and insulin resistance. In clinical trials, it was found that hypoglycemic agents can help AD patients by improving their brain activity. DM is an independent risk factor for cognitive impairment, and its damage to the CNS is getting more and more attention. In recent years, epidemiological studies have shown that DM, especially T2D, increases the risk of AD [88], indicating that DM may play an important role in

the development of AD pathogenesis. A reduction in insulin release and a decrease in insulin levels are critical to the development and progression of both T2D and AD. When insulin signaling is impaired by insulin resistance, it leads to DM, AD and other neurological diseases [89], also the decrease of glucose metabolism in AD patients was observed by 18F-FDG PET technique. Further studies have shown that insulin resistance is associated with a significant reduction in glucose metabolism in the brain of patients with AD, which in turn increased learning and memory dysfunction in AD

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patients [90]. 4.1 Insulin resistance and Aβ deposition

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We already know that GSK-3β plays an important role in insulin resistance caused

by exogenous infection and endogenous lipid metabolism disorders. The close relationship between insulin resistance and Aβ deposition is now described in this paragraph. Insulin resistance may first influence the clearance of normal Aβ by

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affecting mitochondrial function, which in turn affects the clearance of the deposited

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Aβ by inflammatory cascades. Some studies have suggested that Aβ deposition

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involved in immune defense and anti-infection [91], but the formation of an inflammatory microenvironment will break this beneficial mechanism. Under this

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microenvironment, the body will mistakenly believe that a large amount of Aβ is required to be removed as the invasive substance. Interestingly, under normal

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circumstances this will not have too much effect on the body. However, it will eventually lead to a large amount of deposition of Aβ with age, physical dysfunction, and a decrease of Aβ clearance caused by insulin resistance. DM is also considered as

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a chronic mild inflammation-related diseases, and more and more evidence suggests that DM and its complications such as diabetic nephropathy, diabetic retinopathy and diabetic vascular disease and others are closely related to inflammation [92-94]. In

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addition, inflammation in AD plays an important role in the regeneration and apoptosis of hippocampal neurons [95]. Apart from the secretases and lysosomes which are two

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cleavage enzymes for the removal of APP, the erroneously deposited Aβ requires other pathways to be cleared. Experiments have shown that early inflammatory response to activation of microglia-mediated phagocytosis can remove Aβ [96, 97], but sustained and hyperactivated inflammation will further lead to chronic long-term deposition of Aβ, and eventually increase the pathological damage of AD [98]. The inflammatory microenvironment are gradually forming in patients with insulin resistance, and uncontrollable cascade reaction will damage the health of patients step by step. In

conclusion, GSK-3β-involved insulin resistance may aggravate Aβ deposition by multiple routes (Figure 3). 4.2 GSK-3β and tau hyperphosphorylation The traditional viewpoint is that Aβ deposition forms plaques, leading to tau protein hyperphosphorylation, further leading to the death of cells and AD. Lee HG et al challenged this classic concept, suggesting that tau protein phosphorylation is a compensatory response in the process of neuronal antioxidant stress as a protective

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function for neurons [99, 100]. Initial tau phosphorylation may be a protective response,

but excessive phosphorylation of tau will further aggravate the injury, and Aβ will

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attack this protective function until this function is gradually lost. At this stage the level

of toxicity can lead to AD associated neuronal destruction and cognitive impairment. Aβ deposition is toxic to neurons, but the first step of tau phosphorylation is actually to reduce this toxicity. Tau phosphorylation, which is involved in GSK-3β, plays an

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important regulatory role in the brain, but the mutant PS1 may cause GSK-3β

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overexpression, and thus hyperphosphorylated tau which is an important factor in the

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formation of NFTs. In addition to that, insulin resistance and insulin signaling pathways also play an important role in the regulation of tau phosphorylation. In the study of non-

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obese diabetic (NOD) model mice, insulin dysfunction was found to cause AD-like tau hyperphosphorylation in the brain [101]. When insulin was used to treat db/db diabetic

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mice, it was found that it not only restored the phosphorylation levels of insulin receptors and GSK3β, but also improved the learning ability of mice, which is related to reductions in the level of tau phosphorylation in the brain [102]. Kim’s study show

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that moderate exercise can inhibited the hyperphosphorylation of tau in the hippocampus by inhibiting the expression of GSK-3β in diabetic rats and found that the incidence of AD was reduced and that the PI3K/Akt/GSK-3β pathway was involved in

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it [103]. In rats, reducing oxidative stress through the PI3K/Akt/GSK-3β signaling pathway can improve diabetes-induced cognitive impairment [104]. In addition,

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autopsies were performed on patients with DM and/or AD, and the levels and activity of PI3K/Akt signaling pathway were reduced in their frontal cortex, with a large amount of phosphorylated GSK-3β and abnormal tau hyperphosphorylated proteins detected. Interestingly, the defects of this signaling pathway are more severe in individuals who suffer both DM and AD (T2DM-AD) [105]. Excessive activation of GSK-3β is closely related to tau hyperphosphorylation, and inhibition of the GSK-3β over-activation may alleviate tau hyperphosphorylation, thereby slowing the further aggravation of AD

(Figure 3). GSK-3β is not only the important kinases of tau phosphorylation, but also an important kinase of tau hyperphosphorylation, in different status. GSK-3β, as a kinase in insulin signal transduction and tau protein phosphorylation, plays an important role both in the formation and aggravation of insulin resistance, and in the clearance and abnormal deposition of Aβ as well [105]. Therefore, we speculate that GSK-3β is the link between AD and DM. Progress in targeting the GSK-3β in AD

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5.

We have reviewed the important role of GSK-3β in AD and DM, which as a

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potential link between DM and AD, that is a good explanation of why the risk of AD in

diabetic patients more than twice as high as normal [106]. Therefore, a large number of inhibitors for GSK-3β have been studied, and some of the GSK-3β inhibitors have been treated as a therapeutic drug for AD into a Phase II clinical trial [107, 108]. GSK-3β

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inhibitors can be divided into ATP competitive and non-ATP competitive, and the first

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classes of GSK-3β inhibitors were classified as ATP competitive, but non-ATP

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competitive GSK-3β inhibitors with high selectivity [109]. Due to the high selectivity and less toxicity of non-ATP competition, it is considered to be a more promising

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research direction. This section focuses on the progress of GSK-3β inhibitors in AD, and we will review the progress of GSK-3β inhibitors in AD from basic and clinical

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studies (Table 1). First, let's review basic research of GSK-3β inhibitors in AD: in cultured rat cortical cells, the caspase-dependent apoptosis induced by calcineurin inhibitors was prevented by GSK-3β inhibitor 1-Azakenpaullone [110]. And in c-Kit

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Wv/+mice (c-Kit Wv/+ mutation-induced β-cell dysfunction), inhibition of GSK-3β activity by 1-Azakenpaullone improves β-cell function, 1-Azakenpaullone could prevent the onset of diabetes by improving glucose tolerance and β-cell function [111].

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In tauopathies (a group of disorders characterized by aggregation of tau include AD and the fronto-temporal dementias), tau abnormalities significantly disrupt neuronal

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function, before the classical pathological hallmarks of tauopathies. Use lithium and AR-A014418 (another GSK-3β inhibitor) significantly reduced the tau overexpression on axonal, and affect the abnormal behavior of Drosophila [112]. Treatment of primary cultures neurons with lithium and AR-A014418 decreased full-length fractalkine (a large cytokine protein), proving that changing the inflammatory environment may also be the reason for GSK-3β inhibitors's neuroprotective effect [113]. Furthermore the treatment of human neural progenitor cells with IM-12 (GSK-3β inhibitor) resulted in

an increase of neuronal cells, suggest that GSK-3β is closely related to neuronal proliferation [114]. Indirubin and its derivatives have been shown to possess potent inhibitory effects on GSK-3β, use it to treatment APP/PS1 mice, that the spatial memory deficits significantly attenuated, and this was accompanied by a marked decrease in several AD-like phenotypes [115]. In the previous article, we referred to the high fat diet and insulin resistance are closely linked. Treatment of mice (with high fat diet induced cognitive impairment) with Indirubin the learning and memory

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performance of mice increased, suggested that GSK-3β inhibition could prove to be

beneficial in insulin resistance induced cognitive deficit [116]. In addition, Indirubin

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suppresses Aβ-induced apoptosis by inhibiting tau hyperphosphorylation [117] Intrahippocampal TDZD-8 (a non-ATP competitive thiadiazolidine inhibitor of GSK3β) blocked tau hyperphosphorylation and normalized hippocampus-dependent

memory in db/db mice, suggest that the GSK-3β inhibitor plays a protective role in

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T2D-induced memory impairment [118]. Then let's review clinical research GSK-3β

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inhibitors in AD: now the clinical research of GSK-3β inhibitors is still relatively less.

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But as early as 2013, a pilot study of GSK-3β inhibitor tideglusib in AD had been done. Tideglusib produced positive trends in MMSE (Mini-Mental Status Examination),

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ADAS-cog (Alzheimer’s Disease Assessment Scale-cognitive subscale), GDS (Geriatric Depression Scale), and GCA (Global Clinical Assessment) without statistical

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significance in this small sample, this trial provides preliminary evidence to support a benefit of Tideglusib in AD [119]. Then in a randomized trial, Tideglusib reduces progression of brain atrophy in progressive supranuclear palsy, that reduced the

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progression of atrophy in the whole brain, particularly in the parietal and occipital lobes [120]. But in a phase II trial of Tideglusib in AD, many indicators are still no statistical difference, even though BACE1 in CSF significantly decreased with treatment in a

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small subgroup of patients [107]. According to the review earlier in this article, although Tideglusib encountered some obstacles in Phase II clinical trials, we still

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believe that this is a promising target. We think it should be perform PS1 mutation screening in the diabetic patients, because they have a high risk of AD, treatment of this diabetic patients with GSK-3β inhibitors will have a positive effect.

6.

Conclusion Since AD has been discovered, we have never slowed the pace of its exploration,

but AD is still a huge mountain that we are unable to overcome. With the annual incidence of AD increasing dramatically, we urgently need to find drugs that can prevent and/or improve it. Recently it was found that the risk of dementia in DM patients is higher than that of non-diabetic patients and it is also well known that T2D/insulin resistance is involved in AD [121], further evidenced by researchers from

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Rhode Island Hospital and Brown Medical School who discovered that the brain was

also responsible for producing small amounts of insulin and brought up the concept of

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T3D [9], which is a condition where the brain doesn't produce enough insulin or known as the brain diabetes [122, 123]. This breakthrough also attracted more attention to further investigate the link between T2D and AD. There are many complex links between DM and AD, such as neuroinflammation, mitochondrial dysfunction and

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imbalance of intestinal flora, but the key relation is still unclear [98, 124-126]. In recent

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years, tau as a candidate for the treatment of AD has gotten more attention. Tau

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hyperphosphorylation and the resulting formation of neurofibrillary tangles as well as synaptic loss are closely related to memory deficit in patients with AD. Interestingly, a

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large number of recent studies have shown that GSK-3β as a common kinase of insulin signal transduction and tau protein phosphorylation has played an indispensable role in

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DM and AD, which may be the potential link between DM and AD. In DM, GSK-3β plays a key role in the regulation of blood glucose, which is the main rate limiting enzyme for inhibition of glycogen synthesis. More importantly, it is

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one of the key factors leading to insulin deficiency and insulin resistance, and insulin resistance is a hallmark in the occurrence and development of DM. In AD, GSK-3β plays an important role as a kinase in both phosphorylation and hyperphosphorylation

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of tau. Meanwhile insulin resistance in DM may cause Aβ deposition, which will be cleared by tau, but excessive phosphorylation of tau will further aggravate Aβ

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neurotoxicity, damage the brain and affect cognitive function. GSK-3β may not only be a promising therapeutic target, but also an important clue through which we can climb over the mountain of AD. Disclosure statement The authors have no conflicts of interest to disclose. Acknowledgements I gratefully acknowledge my valued colleagues for their contributions to several

studies described in this review. This work was supported by the National Natural Science Foundation of China (81660599, 81360311, 81460548), postgraduate education innovation program of Guizhou Province (11374), the Zunyi Medical University Funds (2013F-686, F-738).

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Figures

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Fig.1. A simplified schematic diagram representing that GSK-3β involved in blood

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glucose regulation.

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Fig.2. A simplified schematic diagram representing the relationship between GSK-3β and insulin resistance.

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Fig.3. A simplified schematic diagram representing the role of GSK-3β in insulin

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resistance-related AD.

Table Table 1 GSK-3β Inhibitors in AD. GSK-3β Inhibitors Inhibitor

Reference(s) [110, 111]

AR-A014418

[112, 113]

IM-12

[114]

Indirubin

[115-117]

lithium

[112, 113]

TDZD-8

[118]

Tideglusib

[107, 108, 119, 120]

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1-Azakenpaullone