Kumju Youn, Chi-Tng Ho, Mir Jun,c,*

a Department of Food Science and Nutrition, Dong-A University, Busan 49315, Korea

b Department of Food Science, Rutgers University, NJ 08901, USA

c Department of Health Sciences, The graduate School of Dong-A University, Busan 49315, Korea

Keywords:

Alzheimer’s disease

β-Amyloid peptide

Green tea

EGCG

Neuroinflammation

A B S T R A C T

Alzheimer’s disease (AD) is the most common neurodegenerative disease characterized by cognitive decline and memory impairment.Many lines of evidence indicate that excessive β-amyloid peptide (Aβ)generation and aggregation play pivotal roles in the initiation of AD, leading to various biochemical alteration including oxidative damage, mitochondrial dysfunction, neuroinflammation, signaling pathway and finally resulting in neuronal death.AD has a complex pathogenic mechanism, and a single-target approach for anti-AD strategy is thus full of challenges.To overcome these limitations, the present study focused to review on one of multiple target-compounds, (-)-epigallocatechin-3-gallate (EGCG) for the prevention and treatment of AD.EGCG is a main bioactive polyphenol in green tea and has been reported to exert potent neuroprotective properties in a wide array of both cellular and animal models in AD.This review demonstrated multiple neuroprotective efficacies of EGCG by focusing on the involvement of Aβ-evoked damage and its Aβ regulation.Furthermore, to understand its mechanism of action on the brain,the permeability of the blood-brain barrier was also discussed.

1.Introduction

Tea is one of the most popular beverages consumed globally.Camellia sinensisis a species of evergreen shrubs belonging to the Theaceae family, the leaves of which are commonly used to produce tea.Depending on the degree of fermentation, tea is categorized into 3 types: non-fermented green tea, semi-fermented oolong tea, and fully fermented black tea.In the case of green tea, steaming or roasting at the first step of production suppresses the enzymatic conversion involved in the oxidation and polymerization of the tea constituents [1].

Green tea maintains the native structure of its polyphenolic compounds as well as its overall composition, while fully fermented tea contains more oxidized polyphenols such as theaflavins and thearubigins.Catechins such as (-)-epigallocatechin-3-gallate(EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate(ECG), and epicatechin (EC) comprise more than two-thirds of all tea polyphenols.EGCG is the most abundant catechin in brewed green tea, accounting for 50%–80% of the total catechins [2-4].

The beneficial physiological effects of green tea have been widely studied, including its antioxidant, anti-inflammatory, antiobesic, anti-diabetic,anticarcinogenic, and anti-neurodegenerative properties [5-9].Recent experimental and epidemiological studies have demonstrated that consumption of green tea is associated with a lower risk of cognitive impairment [10-14].In addition, collective evidence from these studies has revealed that the neuroprotective properties of green tea are mainly attributed to catechins, especially EGCG.The purpose of this review is to provide a comprehensive understanding of the mechanism of neuroprotective action of EGCG in Alzheimer’s disease (AD), mainly focusing onβ-amyloid peptide (Aβ).

2.Alzheimer’s disease

AD, the most common form of dementia, manifests with a gradual onset of irreversible cognitive decline.The pathological hallmarks of AD are extracellular plaques composed of fibrous Aβpeptides and intracellular neuro fibrillary tangles consisting of hyperphosphorylated tau protein.Although the cause of AD is unclear, recent genetic and pathological studies have demonstrated that the deposition of irregular Aβfibrils is a pivotal initiating factor in AD pathogenesis, resulting in various physiological alterations such as tauopathy, oxidative stress, mitochondrial dysfunction, and neuroinflammation [15-17].Since AD is a complex disease, a multifarious, rather than a single approach, is considered more valuable in terms of therapeutics or prevention of the disease.

2.1 APP processing

The proteolytic processing of amyloid precursor protein (APP) is essential for the production of Aβimplicated in AD pathology [18].In the non-amyloidogenic pathway, APP is first cleaved at theα-secretase site to release amino-terminal soluble APP-α(sAPP-α)and generate a carboxyl-terminal fragment (α-CTF or C83),which is indicative ofα-secretase activity [19].In contrast, Aβis produced in the amyloidogenic pathway; the initial cleavage ofβ-secretase (beta-site APP cleaving enzyme I, BACE1) produces an Aβ-containing C-terminal fragment (β-CTF or C99) and a soluble APP-β(sAPP-β) fragment.β-CTF is then cleaved byγ-secretase to generate Aβand a smallerγ-CTF [20].Because of the limited amount of APP, it is considered that amyloidogenic and non-amyloidogenic pathways compete for the substrate in APP proteolysis [21].Thus,the extracellular elevation of sAPP-αcan be inferred as an indirect marker of BACE1 suppression in amyloidogenesis.However,the extracellular secretion of these various fragments can be regulated independently of APP cleavage as well.Therefore,in-depth characterization of the treatment effects on both pathways is needed before conclusive analysis of the underlying mechanisms [22].

2.2 Aβ aggregation

Numerous proteins of diverse sequences, structures, and functions, self-assemble into morphologically similarβ-sheet-rich fibrillar aggregates known as amyloids [23].Abnormal and irregular self-aggregation and misfolding of Aβto a toxic fibrillar amyloid with aβ-sheet structure is considered to be the principal event in the development of AD [24].These fibrils aggregate through a nucleation-dependent pathway composed of two different stages:nucleation and elongation.During nucleation, disordered monomers fold themselves and transform into nuclei.When the connection of the monomers is faster than detachment, and is followed by growth,it is called elongation.However, these oligomersare temporary; they undergo other aggregation steps that generate protofibrils and finally,mature fibrils [25].A characteristic feature ofβ-sheet-rich amyloid fibrils is that they seed suitable structures that can competently convert unpolymerized monomers from the soluble to the aggregated state [26].The main forms of Aβcontain 40 or 42 amino acid residues,commonly identified as Aβ1-40and Aβ1-42, respectively.Because of the strong hydrophobicity of Aβ1-42, it tends to form insoluble fibrils, and is thus more abundant in plaques than Aβ1-40.

2.3 Aβ clearance

Aβlevels in the brain are reduced or eliminated via enzymatic and non-enzymatic mechanisms.In the enzymatic Aβclearance pathway,neutral endopeptidase (NEP) is considered the most compelling Aβ-cleaving enzyme, preferentially degrading Aβ42and Aβ40oligomers [27].NEP is a type II integral membrane zinc metalloprotein that is expressed throughout the brain, primarily on pre- and post-synaptic neuronal membranes [28-30].A previous study reported that both the expression and the activity of NEP decline with age and disease, which may inevitably contribute to Aβaccumulation [31].

The non-enzymatic mechanism of Aβclearance is via Aβtransportation across the blood-brain barrier (BBB) and also via phagocytosis by different cells.In the normal brain, Aβconcentration is regulated through a balance between its production rate and the rate of clearance.The influx of Aβinto the brain across the BBB is mainly through the receptor for advanced glycation end products(RAGE) and its rapid clearance is through the low-density lipoprotein receptor-related protein-1 (LRP-1) [32-34].In AD patients and AD animal models, RAGE expression is increased, whereas LRP-1 expression at the BBB is reduced, thus making it an unfavorable condition for Aβclearance [35-37].The phagocytic activity of neurons, astrocytes, and microglia contributes to Aβclearance to an extent [38].

2.4 Oxidative stress and neuroinflammation

Aggregated Aβfibrils are neurotoxic and spontaneously produce oxidative stress and neuroinflammatory responses.Aβcauses the generation of reactive oxygen species (ROS), including superoxide,hydrogen peroxide, and hydroxyl free radicals, subsequently causing mitochondrial impairment, metal dyshomeostasis, and antioxidant defense system failure, which directly affect synaptic activity and neurotransmission [39].Furthermore, Aβaccelerates neurodegeneration by activating microglia, which exert cytotoxic effects by releasing ROS and RNA [40].Overall, the molecular targets affected by ROS include DNA, RNA, lipids, proteins,cellular architecture, mitochondrial function, receptor trafficking,calcium homeostasis, and energy homeostasis [41].Abnormal and irregular cellular metabolism, in turn, could affect Aβproduction and accumulation and hyperphosphorylated tau, which independently aggravate mitochondrial dysfunction and oxidative stress, thus contributing to a vicious cycle.

Aβstimulates the nuclear factor-kappa B (NF-κB)-dependent pathway, which is a family of inducible transcription factors involved in neuroinflammation [42].NF-κB is found in the cytoplasm as a heterodimeric p50/p65 complex, one of the NF-κB subunits, and is suppressed by an inhibitor of NF-κB (I-κB).Proteasomal degradation of IκB, by IκB kinase (IKK), leads to the release and translocation of NF-κB dimers to the nucleus and binding to the NF-κB response element, which further promotes the expression of target genes,including pro-inflammatory cytokines and inflammatory enzymes,to stimulate oxidative stress and cellular damage [43].Recent studies have shown that the NF-κB signaling pathway plays a crucial physiological role in the central nervous system and plays a critical role in cellular responses to neuronal damage and synaptic plasticity [44].

3.Neuroprotective action and underlying mechanism of EGCG

3.1 Regulation of Aβ deposition and APP processing

One of the key pathological features of AD is Aβaccumulation in the brain.As shown in Table 1, treatment with EGCG was found to reduce the load of amyloid deposits inCaenorhabditis elegans[45].In addition, Aβaggregates were decreased by 60% in the front cortex and 52% in the hippocampus after 3 months of EGCG treatment(20 mg/kg/day) in APP transgenic mouse models [46].These findings were consistent with the results of a study by Lin et al.[47], who showed that 4 months of oral administration of EGCG (20 mg/kg)lowered Aβdeposition in the hippocampus of APP transgenic mice.

Table 1Inhibitory effects of EGCG on the Aβ deposition and APP processing.

Various experiments have thus demonstrated that EGCG regulated APP processing and consequently decreased Aβdeposition.The anti-neurotoxic effect of EGCG via increasing the secreted levels of sAPP-αwas first shown in human SH-SY5Y neuroblastoma and PC12 cells [48].EGCG promoted the non-amyloidogenic process by promotingα-secretase cleavage in Swedish mutant APP overexpressed N2a (SweAPP N2a) cells [49].The results of additional cell-based experiments by Lin et al.[47]were consistent with the previous results, that is, EGCG increased sAPP-αand reduced intracellular Aβformation in MC65 cells.

An EGCG-treated Swedish transgenic mouse model (Tg2576)with intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) routes showed significant reduction in cerebral soluble Aβ1-40and Aβ1-42levels associated with increasedα-secretase activity andα-CTF/sAPP-αproduction [49].In agreement with these findings, chronic oral administration of EGCG (50 mg/kg in drinking water) for 6 months not only reduced Aβlevels and Aβdeposition, but also improved working memory impairment in AD transgenic mice [50].Interestingly, co-treatment of fish oil (8 mg/kg/day) with EGCG(62.5 mg/kg/day or 12.5 mg/kg/day) augmented the production of sAPP-αcompared to either compound alone in Tg2576 mice,implying that these compounds were able to act synergistically on the inhibition of cerebral Aβdeposits [51].

Numerous studies have indicated that the a-disintegrin and metalloprotease (ADAM) family, including ADAM9, ADAM10,and ADAM17, were candidates forα-secretase activation [52].Treatment with EGCG increased the levels of both sAPP-αand ADAM17, known as tumor necrosis factor-α-converting enzyme(TACE), in SweAPP N2a and Tg2576 mice [49].In addition to APP metabolism, ADAM17 was found to influence proteolytic cleavage of other substrates that diminish Aβdirectly or indirectly.ADAM17 also stimulated the shedding of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), fractalkine (FKN), and interleukin(IL)-8, which augment phagocytosis, resulting in Aβdegradation [53,54].Furthermore, ADAM17-dependent stimulation of members of the Epidermal Growth Factor (EGF) family was found to boost neuronal proliferation and reduce cerebral damage in AD [55].Obregon et al.[56]revealed that EGCG increased sAPP-αlevels by promoting the maturation of ADAM10, which was known to be regulated by proprotein convertases, PC7 and furin [57].

The underlying mechanism of EGCG showed that the compound dramatically decreased the levels of both immature and full-length holo-APP by iron chelation, resulting in the inhibition of APP translation [58].Moreover, EGCG upregulated the estrogen receptor-α(ER-α)/PI3K/Akt pathway in SweAPP N2a cells, revealing similarity of its gallate group to 7α-estrogen sites, which allowed EGCG to bind to ER-α[59].Administration of EGCG (2 mg/kg)for a week or two weeks significantly augmented the expression of protein kinase C (PKC) isoenzymesαendεin the hippocampus of mice, which in turn led toα-secretase activation [48].

Severalin vitroandin vivostudies revealed that the decrease inβ- andγ-secretase activity, as well as BACE1 and APP expression,hinted at potential promotion of non-amyloidogenesis via the inhibition of amyloidogenesis [60-65].Jeon et al.[61]used a FRET-based enzyme assay to demonstrate the BACE1 inhibitory property of EGCG.Similar results obtained by Shimmyo et al.[62]indicated that EGCG suppressed Aβ-induced BACE1 upregulation in primary cells of the rat cerebral cortex.Lee et al.[63]reported that sub-chronic administration of EGCG (3 mg/kg) in memory-impaired mice restored Aβ1-42-induced memory dysfunction by decreasing Aβlevels via enhancement ofα-secretase, and the inhibition ofβ- andγ-secretase by way of suppression of the ERK/NF-κB pathway.Lipopolysaccharide (LPS)-induced Aβaccumulation and memory deficiency through decreasedα-secretase and increasedβ- andγ-secretase activities were all regulated by EGCG treatment (3 mg/kg)in an LPS-infused memory deficient mouse model [64,65].

For the prevention of AD, proteolytic cleavage of APP within the Aβdomain byα-secretase is an appealing strategy; this approach has potential neuroprotective effects, because it prohibits Aβgeneration and promotes sAPP-αgeneration [20].Moreover, shedding of the ectodomain is a prerequisite for the cleavage of the intracellular domain ofγ-secretase, a process that releasesα-CTF [66].Therefore,EGCG, with its APP processing (mediated byα-secretase) capability,provides an optimistic and alternative approach for AD prevention.

3.2 Modulation of Aβ aggregation and Aβ clearance

Aggregation ofβ-sheet-rich fibrils, one of the procedures in the amyloid formation cascade, requires a partial unfolding or structural remodeling of the amyloid precursor [67].Such protein misfolding is commonly associated with neuronal toxicity in numerous human diseases, including AD [68].Therefore, one of the promising therapeutic approaches is to inhibit amyloid aggregation and disassemble preformed fibrils.As represented in Table 2, previousin vitrostudies have demonstrated that various synthetic peptides and natural proteins suppress and/or reverse amyloid formation [69,70].However, disassembling large fibrils has been suggested to increase the population of oligomeric amyloid species and exacerbate amyloid toxicity [71].Various studies have shown that EGCG can prevent amyloid aggregation and dissociate fibrils without increasing toxic intermediates [72-76].In addition, the compound has the ability to interact with a wide range of natively unfolded proteins such asα-synuclein (αS), huntingtin (HTT), islet amyloid polypeptide(iAPP), and transthyretin (TTR), and inhibit their aggregation while simultaneously protecting cultured cells against toxicity [77-80].

Table 2Inhibitory effect of EGCG on the aggregation and clearance of Aβ.

The role of EGCG in the inhibition of Aβself-assembly has been widely studied.Based on a two-dimensional magic-angle spinning solid-state NMR study by Lopez del Amo et al.[81], EGCG was found to interact with the C-terminal hydrophobic core of Aβ1-42(i.e., residues 10–20), which was needed to generateβ-sheets for Aβaggregation.Residues 10, 13, and 14 in the hydrophobic core of Aβplayed important roles as potential metal ion coordination centers to generate oxidative stress.In addition, EGCG directly attached to mature Aβfibrils and transformed them into amorphous non-toxic aggregates without disassembling them into monomers or small diffusible oligomers [73-76].On the other hand, Ehrnhoefer et al [82].revealed that EGCG promoted aggregation of Aβmonomers viaan off-pathway mechanism, leading to the formation of unstructured,non-toxic Aβoligomers.

The metal-chelating effect of EGCG plays a critical role in lowering Aβfibril formation.According to the study of surface plasmon resonance imaging (SPRi) by Cheng et al.[83], Aβincubation with metals including Cu2+, Zn2+, and Fe3+triggered strong fibrillar networks of the defined elongated structure, which was attenuated by EGCG treatment viaan off-pathway, producing stable amorphous oligomers without dense fibrillar structures.Similarly,Hyung et al.[84]reported that EGCG disaggregated Aβfibrils in the presence of metals including Cu2+or Zn2+, resulting in the formation of low-molecular-mass and unstructured Aβspecies.Transmission electron microscopy (TEM) and square wave voltammetry (SWV) experiments showed that EGCG interacted with the Aβ-Cu2+complex and exhibited only globular aggregates with non-distinctive edges [85].

Complementary to thein vitroexperiments, computationalin silicosimulation of the structure of EGCG and Aβcomplexes was achieved.According to molecular dynamics (MD) simulation, EGCG expelled H2O molecules from Aβ1-42and directly interacted with Aβ1-42,which was crucial for its inhibitory properties.In addition, it was suggested that both hydrogen and hydrophobic interactions were related to EGCG-Aβinteractions [86].Another study by Wang et al.[87]reported that the major interactions between EGCG and Aβgradually switched from polar to non-polar ones, as the ratio of EGCG/Aβ1-42increased, resulting in a transition of the binding from enthalpydriven to entropy-driven.Specifically, hydrogen bonding occurred in Aβ1-16, while primarily hydrophobic interactions occurred in Aβ17-42at various EGCG concentrations, demonstrating that the balance of hydrogen bonds and hydrophobic interactions ensured the binding of EGCG-Aβover a broad range of solution conditions [88].Moreover,Nguyen and Derreumaux [89]demonstrated that EGCG-Aβ1-42was bound more firmly and sequestered Aβdimers from Aβmonomers.In addition, EGCG induced a structural change in Aβ1-42dimers, including a decrease inβ-sheet content and an increase inα-helical conformation [90].Ahmed et al.[91]demonstrated that EGCG bound to equivalent and independent sites in Aβoligomers and this Aβ-EGCG binding underwent direct tethered contact shifts, which reduced solvent exposure of Aβoligomers, suggesting that EGCG interfered with the secondary nucleation process, generating Aβdamage.Recent MD simulations by Zhan et al.[92]demonstrated that EGCG changed the structure of the Aβ1-42proto fibril by disrupting the hydrogen bonding between H6 and E11.In addition, the central core region of the Aβ1-40fibrils was found to possess a strong affinity for EGCG because of its hydrophobic residues [93].

Regarding Aβdegradation and clearance, EGCG has been shown to reduce Aβlevels via protease-mediated Aβregulation.Melzig and Janka [94]reported that EGCG elevated extracellular NEP levels and regulated Aβmetabolism by increasing NEP activity and activating the ERK/PI3K pathway in astrocytes.However, other catechins,including EC, EGC, and ECG, had no effect on NEP secretion [95].Similar results were obtained fromin vitroandin vivoAD models, that is, EGCG recovered both cognitive impairment and Aβaccumulation by upregulating NEP expressionvia suppression of histone deacetylase 1(HDAC1), a negative regulator of the NEP gene [96].

3.3 Suppression of neuroinflammation and oxidative stress

EGCG attenuated Aβ-induced oxidative stress and mitochondrial impairment in several neuronal cell lines and AD mouse models(Table 3).The compound inhibited Aβ-induced damage by reducing ROS production and lipid peroxidation in cultured hippocampal neurons [97].In addition, EGCG augmented neuroprotection against Aβ-mediated oxidative stress by enhancing the antioxidant defense system, mainly glutathione (GSH) biosynthesis in BV2 microglial cells [98].Chronic oral administration of EGCG-rich green tea extract for 26 weeks markedly lowered both lipid peroxides in plasma and ROS concentration in the hippocampus of Wistar rats [11].In addition, orally administered EGCG (10 mg/mL in drinking water for 5.5 months) not only attenuated ROS production, but also restored mitochondrial function, respiratory rates, mitochondrial membrane potential (MMP), and ATP levels in APP/presenilin 1 (PS1) double transgenic mouse models [99].

Table 3Inhibitory effects of EGCG on Aβ-induced oxidative stress and neuroinflammation.

In vitrostudies based on the anti-neuroinflammatory properties of EGCG against Aβhave been performed on different cell lines,including PC12, U373MG, EOC 13.31, N2a/APP695, and BV2 cells[100-103].EGCG diminished the activation of the NF-κB pathway,consequently preventing the production of cytokines and vascular endothelial growth factor (VEGF) in human astrocytoma U373MG cells [100].Wei et al.[101]indicated that EGCG suppressed Aβstimulated neuroinflammatory responses of microglia, such as TNFα,IL-1β, IL-6, and iNOS, and improved the levels of intracellular antioxidants including Nrf2 and HO-1 in EOC 13.31 microglia.The compound suppressed Aβ-induced NF-κB stimulation and mitogen-activated protein kinase (MAPK) signaling, including c-Jun N-terminal kinase (JNK) and p38 signaling.Furthermore, EGCG inhibited oxidative stress and inflammatory markers through the upregulation of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in N2a/APP695 cells [102].A recent study by Zhong et al.[103]demonstrated that EGCG suppressed the activity of canonical and non-canonical inflammasomes through the toll-like receptor 4 (TLR4)/NF-κB pathway in BV2 microglial cells stimulated by Aβexposure bothin vitroand in APP/PS1 mice, implying that EGCG downregulated inflammation and neurotoxicity in microglia.

Table 4Bioavailability and BBB permeability of EGCG.

3.4 BBB

The absorption and pharmacokinetics of EGCG in various brain sections have been revealed by oral and intravenous administration of the compound, suggesting that EGCG potentially penetrated the BBB in brain tissue [110].Anin vitroBBB permeability study demonstrated that 5% of the EGCG reached the brain parenchyma in 1 h; the study revealed that BBB permeability was suppressed by EGC and that of EGC was further suppressed by GA, implying that ingested catechins can be controlled by coexistence of the other ones [111].Interestingly, after oral administration of EGCG (100 mg/kg/day),the compound was not found in the brain tissues of young rats, but was identified in aging rats with cognitive impairment (CI).The increase in BBB permeability in aging rats with CI suggests that the BBB permeability change could act as a structural basis for EGCG treatment in memory and learning improvement [112].

4.Summary

The present review summarizes the molecular mechanisms underlying the neuroprotective action of EGCG in AD as follows:1) inhibition of Aβdeposition by regulating APP processing,2) preventing Aβaggregation and dissociating Aβfibrils,3) promoting enzyme-mediated Aβdegradation, 4) attenuating Aβinduced oxidative stress, and 5) reducing neuroinflammatory response induced by Aβ(Fig.1).The BBB permeability of intact EGCG implies that the compound might be a powerful source to prevent AD,rendering a strong physiological basis for the improvement of various cognitive diseases.Despite the encouraging results obtained from previous preclinical studies, a translational gap exists between basic discovery and clinical application.Further in-depth research should be explored and human clinical trials need to be conducted to establish the neuroprotective efficacy of EGCG and leverage it successfully in AD therapeutics.

Fig. 1 The molecular mechanisms of neuroprotection by EGCG.

The BBB is a selective diffusion barrier that blocks some substances in the blood from entering the brain tissue, in order to maintain a constant internal environment.The ability of a substance to cross the BBB is an essential prerequisite for it to exert a neuroprotective effect in the brain.EGCG, after consumption, was found to hydrolyze to gallic acid and EGC in the intestinal microbial environment (Table 4) [104].While most EGCG and its metabolites were excreted in the bile, a portion of intact EGCG was found to enter the blood circulation, peaking at approximately 1-2 h after administration in rats and humans [104-108].A study with labeled[3H](-)EGCG by Suganuma et al.[109]demonstrated that EGCG was easily absorbed and distributed in various organs, including the brain, and a small amount was excreted in the urine after direct oral administration.In addition, the duplicated administration of[3H](-)EGCG increased the radioactivity by approximately 6 times in the blood, a less dramatic increase was observed in the digestive tract, and a distinctive increase of radioactivity was seen in the brain by approximately 6.8 times, implying that frequent green tea consumption might enable the distribution of high levels of polyphenols in various organs [109].

Declaration of competing interest

The authors declare no conflict of interests.

Acknowledgments

This research was supported by Dong-A University.