Green Tea Epigallocatechin-3-gallate (EGCG) Targeting Protein Misfolding in Drug Discovery For Neurodegenerative Diseases Part 3

4.2. Evidence from Animal Models

Because of its broad spectrum of pharmacological activities, EGCG displayed therapeutic potential on various in vivo models of NDs, including AD, PD, HD, and ALS (Table 2). 

EGCG is a compound that is widely found in tea leaves, especially green tea. It has been widely studied and has been shown to have many health benefits, including improved memory. This is a very positive and beneficial finding, as many people face memory problems as the population ages.

Studies have shown that EGCG can help improve memory. EGCG can protect nerve cells by reducing fragility and oxidation. It can also stimulate the growth and connection of neurons, improve neurotransmission, and enhance cognitive function in the brain. These processes help improve cognitive abilities such as long-term memory and attention.

In addition, EGCG can also increase blood flow in the brain, providing more oxygen and nutrients, which helps protect nerve cells and improve brain function.

In summary, EGCG is positive and beneficial for improving memory. It can promote memory and cognitive ability through a variety of pathways and improve brain health and function. Therefore, drinking green tea in moderation can enable people to gain many health benefits and improve their quality of life. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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The therapeutic potential of EGCG in an animal model of AD was first reported in 2005 using Swedish mutant APP-overexpressing mice (Tg APPsw). The researchers demonstrated that intraperitoneal (i.p.) injection (20 mg/kg) of EGCG significantly decreased both Aβ levels and Aβ plaques in the brain. 

The study further demonstrated that EGCG promoted cleavage in the α-C-terminal fragment of APP and elevated the N-terminal APP cleavage product, soluble APP-α. 

This suggests that the reduction of cerebral Aβ levels is associated with increased α-secretase cleavage activity [184]. Moreover, EGCG similarly reduced Aβ deposition in Tg APPsw when administered orally in drinking water (50 mg/kg), as observed in a study conducted in 2008. The results further indicated that EGCG provides cognitive benefits and modulates tau hyperphosphorylation in these AD transgenic mice [186]. 

Another study involving Tg APPsw mice focused on the molecular mechanism of neuroprotective action of EGCG. EGCG was found to reduce Aβ-induced neurotoxicity by inhibiting glycogen synthase kinase-3β (GSK-3β) activation and c-Ab/FE65 complex nuclear translocation in these transgenic mice [177]. 

In addition to transgenic mouse models of human AD pathology, toxin-induced models are also considered to be suitable for exploring the treatments of NDs. 

The neuroprotective effects of EGCG have been reported in toxin-induced AD models such as Aβ-induced, LPS-induced, D-galactose (D-gal)-induced, streptozotocin-induced, and aluminum chloride (AlCl3)-induced AD models [13]. Two studies from the 2000s and early 2010s used the LPS-induced AD model with orally administered EGCG (1.5 or 3 mg/kg). 

They demonstrated that EGCG prevents apoptotic cell death by preventing elevated levels of Aβ via the inhibition of β- and γ-secretases. The findings also showed that EGCG prevents memory impairment and amyloidogenesis by inhibiting neuroinflammatory-related cytokines released from astrocytes [187,190]. 

The oral administration of EGCG (1.5 or 3 mg/kg) has also been shown to protect against Aβ-induced memory and coordination impairment in Aβ-induced AD rats, while intragastrical (i.g.) administration of EGCG (2 or 6 mg/kg) had potent neuroprotective effects on aging mice induced by D-gal, acting via antioxidative and antiapoptotic mechanisms [188,189]. 

Furthermore, additional evidence from toxin-induced models confirms the potential of EGCG to improve oxidative stress with streptozotocin-induced and AlCl3-induced models. 

These studies showed that EGCG can reduce oxidative stress in peripheral and brain tissue and that it may suppress behavioral changes related to toxin-induced cognitive deficits in these animal models [191,195]. The senescence-accelerated mouse prone (SAMP8), a spontaneous animal model of accelerated aging, has also been used in studies with EGCG. 

This animal model is considered a robust model for studying the pathology of sporadic AD [225]. Studies have demonstrated the ability of EGCG to attenuate cognitive deterioration and memory deficits in these mice via the administration of low (5 mg/kg) and high doses (15 mg/kg) [193,194]. EGCG administration was shown to upregulate the anti-apoptotic protein Bcl-2 and amplify the Bcl- 2/Bax ratio and an association was found between EGCG-induced reduction in Aβ accumulation and elevated neprilysin expression (the rate-limiting degradation enzyme of Aβ) [193]. 

In addition to upregulating neprilysin expression, EGCG was shown to inhibit BACE-1 activity and ameliorate abnormal synaptic protein levels in the frontal cortex and hippocampus in the SAMP8 mice model [194]. 

Returning to the transgenic mice model, in the late 2010s, studies with APP/PS1 mice provide additional evidence for the in vivo neuroprotective properties of EGCG. One study investigated the effect of EGCG on neuronal apoptosis induced by endoplasmic reticulum (ER) stress, revealing that EGCG treatment inhibited ER-stress-induced apoptosis in the cerebral cortex of APP/PS1 mice [196]. 

It has also been suggested that therapies combining EGCG and ferulic acid in aged APP/PS1 mice confer additional benefits over single treatments in terms of improving behavioral deficits, ameliorating cerebral amyloidosis, and reducing Aβ generation [197]. There is also evidence that dual-drug loaded nanoparticles of EGCG/ascorbic acid enhance the therapeutic efficacy of EGCG in APPswe/PS1 transgenic mice [198]. 

Recently, a study with a well-established preclinical mixed model of familial AD and type 2 diabetes mellitus (T2DM) using transgenic APP/PS1 mice fed with a high-fat diet revealed that EGCG improves cognitive deficits aggravated by an obesogenic diet through modulation of the unfolded protein response [199]. EGCG has also been widely studied in toxin-induced animal models of PD, including the classical 6-OHDA and MPTP, which are the two most extensively used neurotoxins for in vivo PD models [226]. 

The neuroprotective effect of EGCG on the MPTP-induced PD model was first reported in the early 2000s. Oral administration of EGCG (2 and 10 mg/kg), as well as green tea extract (0.5 and 1 mg/kg) containing high levels of EGCG, clearly alleviated dopamine neuron loss in the substantia nigra and TH protein level depletion [201]. In this model, the toxicity of MPTP is mediated by oxidative stress, especially by NO. 

Another study tested whether EGCG attenuates MPTP-induced PD in mice by inhibiting neuronal NO synthase (nNOS) expression. The outcomes revealed that both green tea extract and EGCG decreased expressions of nNOS in the substantia nigra [202]. In the 2010s, additional studies using the MPTP-induced PD model confirmed the neuroprotective effect of EGCG against MPTP neurotoxicity [204,205]. 

EGCG was shown to regulate the iron-export protein ferroportin in substantia nigra, reducing oxidative stress and exerting a neurorescue effect against MPTP-induced functional and neurochemical deficits in mice [204]. A 2018 study with an MPTP-induced mouse model of PD focused on the effects of EGCG on the peripheral immune system. 

The outcomes revealed that EGCG treatment protects dopaminergic neurons from MPTP-induced degeneration, restoring the movement behavior of these mice. In addition, EGCG inhibited the expression of neuroinflammatory cytokines and reversed the T-cell dysfunction in the peripheral immune system of MPTP mice [205]. 

An in vivo study with a 6-OHDA-induced PD rat model showed that EGCG administered by gavage (10 mg/kg) reverts the striatal oxidative stress and immunohistochemistry alterations. Furthermore, EGCG treatment attenuated the behavioral changes, indicating neuroprotection manifested as decreased rotational behavior, increased locomotor activity, anti-depressive effects, and improvement of cognitive dysfunction [203]. 

Recently, another study with a PD rat model focused on the effects of EGCG on ROT-induced motor and neurochemical dysfunctions. This study demonstrated that the possible neuroprotective effects of EGCG (100 or 300 mg/kg i.p.) against ROT-induced motor and neurochemical dysfunctions in rats are associated with its antioxidant effects, prevention of mitochondrial dysfunction, prevention of neurochemical deficiency, antineuroinflammatory effects, and anti-apoptotic effects [207]. 

EGCG supplementation was recently shown to result in profound changes in gut microbial compositions in an invertebrate PD model, restoring the abundance of a set of bacteria [206]. The study with PTEN-induced putative kinase 1 (PINK1) mutant flies showed that EGCG ameliorates neuronal and behavioral defects by remodeling gut microbiota [206]. 

In PD animal models, gut microbiota regulates motor deficits and neuroinflammation [227]. Interestingly, emerging evidence suggests that EGCG remodels the architecture of human gut microbiota [228,229]. 

However, further studies are necessary to state that the modulation of gut microbiota is related to EGCG's mode of action. Researchers are now convinced that gut bacteria may be involved in many NDs, and remodeling the gut microbiota to maintain their balance might be a novel therapeutic strategy [230,231]. Indeed, the most recent drug approved for AD was sodium oligomannate, whose mode of action is related to gut microbiota and neuroinflammation, highlighting the importance of the gut microbiome as a potential drug target for AD [53]. 

The wealth of evidence from studies with animal AD and PD models suggests that EGCG may be useful for treating AD and PD. However, the available evidence of EGCG neuroprotective effects is not just limited to studies with AD and PD animal models. 

EGCG has also demonstrated in vivo potential in preventing other NDs in studies published since the 2000s, including transgenic mouse models of ALS and FAP, a Drosophila model of HD, and a 3-nitro propionic acid-induced rat model of HD [25,208–211]. Taken together, these preclinical animal studies suggest that EGCG has considerable potential as a drug candidate for neurodegenerative drug discovery.

4.3. Evidence from Clinical Trials

The efficacy of EGCG has been demonstrated in preclinical models of NDs in the 2000s and 2010s; thus, it was approved for a clinical trial in 2019. To this end, a randomized, double-blind, placebo-controlled parallel-group phase III study was conducted in patients with multiple system atrophy (MSA), a rare ND characterized by aggregation of α-syn in oligodendrocytes and neurons, which shares neuropathological features with PD. 

However, this trial found that supplementation of EGCG did not affect MSA progression. The same study found that although EGCG was generally well tolerated, it was associated with hepatotoxic effects in some patients, thus doses of more than 1200 mg should not be used [232]. There are also reports that phase II clinical trials for AD (NCT00951834) and HD (NCT01357681) have been completed. 

Given the role of oxidative stress in the pathogenesis of NDs, antioxidant therapy for NDs has received considerable attention in recent years as a promising approach to delay or slow oxidative stress-induced neurodegeneration. Many antioxidants, including EGCG, have undergone clinical studies in recent years. Nevertheless, there are barriers to taking antioxidants from the preclinical stage to clinical settings, which are mainly related to pharmacokinetic and pharmacodynamic constraints. 

Hence, despite promising outcomes in vitro and in vivo assays, few to no positive results have been attained to date in clinical trials for any of the antioxidants investigated [233]. 

Researchers have assumed that the low bioavailability of EGCG was an important factor behind these inconsistencies. Indeed, it has been suggested that the therapeutic potential of EGCG is limited by its poor systemic absorption following oral administration, including low absorption, poor pharmacokinetics and bioavailability, scarce biodistribution, first-pass metabolism, trivial penetration, and low accumulation in the related tissues of the body, or low targeting efficacy. 

Furthermore, EGCG is unstable under physiologic conditions, and it can be rapidly degraded or metabolized due to interactions with the hydroxyl groups on the phenol rings. Intravenous administration of EGCG results in partial degradation before it reaches the target tissues [234].

5. EGCG Targeting Misfolded Aggregates in AD and PD

The in vitro effects of NPs that target protein misfolding have been widely studied by numerous independent research groups, combining biochemical and biophysical characterizations with imaging techniques and cell viability assays. 

These included transmission electron microscopy (TEM), atomic force microscopy (AFM), thioflavin (ThT) binding assay, dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, small-angle x-ray scattering (SAXS), and nuclear magnetic resonance (NMR) [125]. 

Multiple studies have shown the effectiveness of EGCG at inhibiting Aβ and α-syn aggregation and other proteins (Table 4). EGCG was first reported in 2006 by Dagmar E. Ehrnhoefer et al. as a modulator of early events in huntingtin misfolding, which changes the conformation of the huntingtin and prevents the formation of destructive protein-protein interactions [25]. 

Next, Ehrnhoefer et al. turned to investigate whether EGCG has the same effect in the fibrillogenesis of other aggregation-prone 'natively unfolded' polypeptides such as Aβ and α-syn. 

They were able to show that EGCG effectively inhibited the fibrillogenesis of both α-syn and Aβ by directly binding to the natively unfolded polypeptides, which can also prevent their conversion into toxic, on-pathway aggregation intermediates. This suggests that EGCG recognizes unfolded polypeptides and directly binds to the main chain common to all proteins [26].

This discovery suggests that EGCG could potentially be a candidate for developing a common pharmacological therapy for NDs and protein misfolding and amyloid diseases in general. Since these findings became known, many studies postulated that EGCG can reduce the neurotoxicity of misfolded proteins related to numerous NDs. 

Most studies in this area have been focused on Aβ and α-syn aggregation and toxicity (Table 4). In the early 2010s, after findings demonstrated that EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers, researchers investigated whether EGCG disassembles preformed amyloid fibrils. 

The results revealed that the EGCG treatment of amyloid fibrils does not reverse the amyloid formation process but rather directly converts fibrillar species into benign protein aggregates, suggesting that EGCG is a potent remodeling agent of mature amyloid fibrils [148]. Researchers found that EGCG binds to preformed Aβ and α-syn amyloid fibrils and oligomers and directly alters their morphology, as confirmed by biochemical, biophysical, and cell-based assays, providing experimental evidence that EGCG directly binds to βsheet-rich aggregates, mediating a conformational change without disassembling them into monomers or small diffusible oligomers [148]. 

It was proposed that a compound-mediated reorientation of bonds between ordered protein molecules in the polymer might be responsible for amyloid remodeling and the appearance of unordered, amorphous protein aggregates. 

On the other hand, another study has suggested that EGCG can robustly disaggregate pre-formed oligomers and has a potent dose-dependent inhibitory activity on α-syn aggregation. This study used confocal single-molecule fluorescence spectroscopy to characterize the effects on α-syn oligomer formation of 14 phenolic compounds and black tea extract, including EGCG. 

The structure-activity analysis concluded that the presence of three vicinal groups on a single phenyl ring, which has been observed in compounds such as EGCG, can be an important molecular feature for more effective in inhibiting and destabilizing self-assembly by α-syn [212]. 

Concerning the chemical mechanisms of EGCG–Aβ interactions, one study showed that EGCG interferes with the aromatic hydrophobic core of Aβ, forming nontoxic Aβ oligomers. Using magic angle spinning solid-state NMR investigations, researchers demonstrated that the nontoxic aggregates formed in the presence of EGCG are well structured, with a characteristic hairpin structure commonly observed for Aβ fibrils and oligomers [128]. 

A study aimed at understanding the molecular mechanism by which EGCG remodels mature amyloid fibrils was published in 2013 and showed that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG [213]. 

EGCG can self-oxidize, affording a complex mixture of monomeric and polymeric EGCG-based quinones, and autooxidation has been considered one of the major reactions causing the in vitro instability of EGCG [235]. 

Researchers have suggested that the molecular mechanism by which EGCG remodels mature amyloid fibrils may be partially due to hydrophobic binding and Schiff base formation with lysine residues of proteins. It was hypothesized that aberrant post-translational modifications mediated by EGCG treatment could occur concomitantly with the hydrophobic remodeling process that seems to be the main driver for EGCG amyloid remodeling. 

Finally, the primary hydrophobic binding mechanism suggested for amyloid remodeling by EGCG and, more likely, its oxidation products may also explain the ability of EGCG to prevent the fibrillar aggregation of monomeric amyloidogenic proteins, perhaps by binding to and making oligomeric seeds less kinetically competent [213]. 

Additionally, another study from 2013 noted that oxidized EGCG can be useful for fabricating drug-delivery microparticles from naturally reproducible and edible green tea [236]. 

More recently, it was demonstrated that green tea polyphenol microparticles based on the oxidative coupling of EGCG inhibit amyloid aggregation/cytotoxicity of the protein α-syn and serve as a platform for drug delivery [219]. The study proposed using EGCG microparticles as a possible bifunctional strategy, blocking amyloidogenesis directly and carrying a molecule that can act synergistically to potentiate the anti-amyloidogenic effect [219].

In 2014, a study that combined NMR spectroscopy, TEM, and CD characterized the interaction of green tea catechins (epicatechin, epigallocatechin, epicatechin gallate, and EGCG) with toxic oligomers in detail.

The flavan-3-ol unit of catechins was shown to be essential for EGCG–Aβ interactions. In addition, similar experiments still demonstrated that the same flavan-3-ol unit is also essential for the interaction with oligomers of PrP [214]. The effect of EGCG on tau aggregation has also been reported. 

This effect was characterized in a study conducted in 2015, which combined cell-free aggregation and a cell-based assay, and found that EGCG could be a potent inhibitor of tau aggregation and toxicity, preventing the aggregation of tau protein into toxic oligomers at substoichiometric ratios [150]. 

More recently, a study with various biophysical and biochemical analyses suggested a possible dual effect of EGCG on aggregation inhibition and disassembly of full-length tau and their binding affinity [28]. 

Returning to studies of α-syn oligomers, it has been proposed that the EGCG's mechanism of action to inhibit the formation and block the toxicity of oligomers is related to EGCG affecting the degree of binding of α-syn oligomers to membranes, resulting in the reduction (but not complete elimination) of the oligomer's affinity for cell membranes. 

According to in vitro assays, this does not affect oligomer size distribution or secondary structure. The data demonstrated that EGCG immobilizes the C-terminal region and moderately reduces the binding degree of oligomers to membranes [149]. 

It has been recognized that EGCG does not inhibit α-syn fibril formation but rather exerts protection against α-syn cytotoxicity, both by enriching the population of nontoxic off-pathway oligomers as well as by promoting the conversion of toxic oligomers into the less toxic fibril (Figure 4). 

This might be attributed to EGCG binding to on-pathway oligomers and accelerating their conversion to fibrils so that the effective population of toxic oligomeric intermediates can be reduced [131].

In this regard, a 2017 study by Yang et al. provided convincing evidence that EGCG does not inhibit α-syn fibrillation but facilitates it, contradicting previous reports that EGCG acts as a remodeling agent favoring the production of small amorphous protein aggregates from mature fibrils [26,131]. 

A sample of a co-incubated mixture of α-syn with EGCG completely diminished the fluorescence signal in a ThT binding assay, indicating a possible full inhibition of amyloid formation. 

Morphologically altered fibrils which appeared slightly curly compared to the straight fibrils obtained in the same fibrillation conditions but in the absence of EGCG were observed. The authors thus suggested that the ThT binding assay is inappropriate to evaluate the 'inhibitory' effect of EGCG on α-syn fibrillation [131].

Although ThT is a gold standard probe to detect amyloid fibrils in vitro, it has been recommended caution to test polyphenols using the ThT binding assay since some critical limitations have been reported such as background signal and competitive binding with the extrinsic probe. This method needs to be validated by other techniques to confirm that the significant decrease in ThT fluorescence observed in the presence of the evaluated compound is related to an inhibitory effect on amyloid fibrillation or rather by compound displacement by ThT [237–240]. 

It should be mentioned that the reduction in the emission in the presence of EGCG might result from the competition of this compound with ThT for amyloid fibril binding sites [237,238]. Recently, pentameric thiophene fluorescence was proposed as an alternative probe to monitor the aggregation kinetics in the presence of EGCG [240]. According to the study by Yang et al., it is pertinent to consider that EGCG induces two types of αsyn oligomers. The TEM and AFM findings revealed that EGCG-treated α-syn not only produces the off-pathway compact oligomers but also produces amyloid fibrils formed by the conversion of on-pathway toxic oligomers into fibrils. 

This supports the hypothesis that EGCG acts to facilitate the conversion of on-pathway oligomers into amyloid fibrils, suppressing this population of toxic oligomers [131]. In a recent study, researchers confirmed that EGCG can accelerate α-syn amyloid fibril formation by facilitating its heterogeneous primary nucleation. 

This work showed that the aggregation conditions define whether EGCG is an inhibitor or enhancer of α-syn amyloid fibril formation since the inhibitory action is not robust against various physiologically relevant changes in experimental conditions [241]. Taken together, these last findings suggest that EGCG can exert its neuroprotective effect against α-syn-induced cytotoxicity by modifying the aggregation pathway toward the formation of nontoxic aggregates and ameliorating oligomer-induced toxicity, possibly through reducing the extent of cell membrane permeabilization-induced by toxic aggregates [131,149]. In another study published in 2017, the effects of EGCG on the fibrillation and disaggregation of α-syn at a molecular level were assessed by using chemical, biochemical, and cell-based methods like ThT binding assay, NMR, microscopy, and MTT. 

These studies indicated that EGCG binding to Leu, His, Phe, and Tyr amino acid residues inhibits the conformational transition of α-syn to β-sheet-enriched conformers and also disaggregates the amyloid fibrils of α-syn in a dose-dependent manner [151]. Additionally, in 2017, a study compared the effect of EGCG on α-syn aggregation to other three inhibitors (dopamine, amphotericin-B, and quinacrine dihydrochloride). The experiment conducted in this study revealed that, at high concentrations, EGCG slows down fibrillization kinetics. 

EGCG was also able to reduce the toxicity of α-syn aggregates in a concentration-dependent manner. The morphological study of nontoxic aggregates formed in the presence of EGCG showed a smaller fibrillar size. Researchers proposed that the decrease in cytotoxicity of α-syn aggregates in the presence of a higher concentration of EGCG can be attributed to its ability to reduce the exposure of a hydrophobic surface, as shown in 8-anilino-1-naphthalenesulfonic acid (ANS) binding studies [215]. 

Interestingly, the ANS binding assay has already provided valuable insights into the role of exposure of hydrophobic surfaces as a result of the aggregation of misfolded species, which is a crucial and common feature of these pathogenic species [242]. It is now known that the exposure of hydrophobic groups on the oligomer surface appears to be a major determinant of oligomer-mediated toxicity. A range of proteins, oligomeric species of similar sizes and morphologies but with very different toxicities, have been isolated and shown to differ in their solvent-exposed hydrophobicity [243]. 

There is also strong evidence that Aβ toxicity is regulated by the solvent exposure of hydrophobic surfaces. Meanwhile, the exposure of hydrophobic surfaces is the decisive factor for toxicity rather than the presence of β-sheets. Multiple lines of evidence suggest that exposure to these toxic surfaces facilitates interactions with multiple cellular components, including membranes, which underlie key pathogenic steps in AD progression [244,245].

Since the exposure of hydrophobic surfaces seems to remodel misfolded protein aggregation, recent studies reported that the mechanism of action of EGCG against protein misfolding is directly related to the regulation of the solvent exposure of hydrophobic surfaces. EGCG, therefore, reduces the toxicity of misfolded aggregates by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure [216,217]. 

Ahmed et al., in 2017, elucidated how EGCG remodels Aβ oligomers using 15N and 1H dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, DLS, and electron microscopy. The experimental findings suggested that the mechanism of amyloid inhibition of EGCG is driven by the preferential binding to Aβ oligomers [216]. The solution NMR studies concluded that EGCG can bind weakly to Aβ monomers while it displays a higher affinity toward oligomers. 

It is speculated that the Aβ40 oligomers become less exposed to solvent upon binding to EGCG, and the β-regions, which are involved in direct monomer−protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift [216]. To gain further insight into the structural determinants of Aβ toxicity, Ahmed et al. investigated Aβ oligomers with different toxicity degrees generated in the absence or presence of a catechin library that included EGCG and six EGCG analogs. 

By combining cell toxicity assays, electron microscopy, NMR spectroscopy, DLS, wide-angle X-ray diffraction (WAXD), and fluorescence assays, the comparative analysis identified a cluster of key toxicity determinants and the associated mechanism of action, which includes exposure of a hydrophobic surface spanning residues 17–28 and the concurrent shielding of the highly charged N-terminus [217]. 

The study findings indicated that toxic Aβ assemblies (Aβn) exhibit solvent-exposed hydrophobic sites accessible to ANS binding, while EGCG-remodeled Aβ aggregates are less toxic and have fewer exposed hydrophobic sites. These exposed hydrophobic surfaces facilitate the colocalization, interaction, and subsequent insertion of Aβ assemblies into the membrane. 

These findings indicate that Aβn toxicity is regulated by the solvent exposure of hydrophobic surfaces [217]. Up to this point of our discussion, all the in vitro evidence strongly suggests that EGCG targets protein misfolding, indicating a potential therapeutic for NDs. 

However, it is important to recognize that all cited studies have analyzed amyloid fibrils and other aggregates that are formed from chemically synthetic or recombinantly expressed peptides or proteins in vitro, and in vitro amyloid fibril structures may differ profoundly from ex vivo amyloid fibril structures [246,247]. It is unclear whether the assays using protein structures that may not represent the in vivo amyloid structure can truly explain how EGCG acts against protein misfolding in vivo.

6. Conclusions

A considerable body of evidence that supports the use of EGCG for ND therapy has become available over the past two decades. The potential effects of EGCG in NDs are now well described and proven by a range of experimental in vitro and in vivo assays discussed in this review. 

However, despite extensive evidence from in vitro and in vivo models of AD, PD, and other NDs suggesting that EGCG is a promising drug candidate for ND treatment, clinical evidence on its anti-neurodegenerative effects does not yet exist. In a clinical trial phase III with MSA patients, no effect on disease progression was found, while some patients showed hepatotoxic effects [232]. 

Some authors have suggested that the inconsistency between the evidence from preclinical stages and clinical trials in humans might be attributed to catechins, like EGCG having poor pharmacokinetic properties and bioavailability, limiting their effectiveness as drug leads. However, this could be improved through new techniques such as nanoparticle-based delivery systems, structurally modified molecules of catechins, or co-administration with other drugs or bioactive compounds [234]. 

The ability of a small molecule to reduce the toxicity of oligomeric species has been shown to represent a promising therapeutic strategy against NDs. EGCG has been shown to significantly reduce the cell toxicity of several amyloid aggregates by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure. 

These findings suggest that the use of EGCG could, therefore, be viewed as a common drug therapy for many NDs targeting protein misfolding, a common feature of numerous NDs. It is also important to note that the studies of the mechanisms by which EGCG combats neurodegeneration have already provided significant advances in our knowledge of the mechanisms of protein misfolding toxicity. 

Furthermore, EGCG and other green tea catechins have been recognized as valuable tools for identifying the drivers of amyloid aggregation and developing other aggregation modulators through structural mimicry [16]. 

Notably, a deeper understanding of the molecular mechanisms of the interactions between small molecules and a misfolded protein may have a prominent role in rational drug design and the development of new therapeutic strategies for NDs. The ultimate message that emerges is that, although current EGCG research has limited impact on clinical practice, it has provided strong evidence and testable hypotheses to contribute to clinical advances and neurodegenerative disease drug discovery.

Author Contributions: P.B.G. conceived the topic, conceptualized the design, and wrote the manuscript. A.C.R.S. and Y.C. reviewed the manuscript and contributed to the design of the study. All authors have read and agreed to the published version of the manuscript.

Funding: This work was funded by the National Council for Scientific and Technological Development (CNPq); National Council for the Improvement of Higher Education (CAPES) Finance code 001; and Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State (FAPERJ).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Acknowledgments: The authors acknowledge Lucas Machado Ascari and Mariana J. do Amaral for providing helpful discussions.

Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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