Mechanism of EGCG intervention
Dr. Jan Bieschke
Diseases of protein misfolding
Many diseases that occur mostly with age are caused by protein misfolding. These include the most common neurodegenerative disorders Alzheimer's and Parkinson's diseases as well as systemic disorders in which the peripheral organs are affected. For example, this is the case for AL and AA amyloidoses and adult-onset (type II) diabetes. These disorders all have in common that a protein accumulates in an insoluble form in the affected tissue. The location of the deposits and the identity of the proteins are characteristic for each particular disorder (Dobson 2003).
Cause-oriented strategies for intervention in amyloidosis disorders
Therapeutic approaches to Alzheimer's or Parkinson's disease are for the most part still directed towards treating the symptoms. Cause-oriented approaches are currently in the developmental stage, i.e. under clinical or preclinical evaluation (Dasilva and McLaurin 2004; Jakob-Roetne and Jacobsen 2009).
The pharmacological intervention strategies described here are oriented primarily towards the process of protein misfolding and amyloid formation occurring in Alzheimer's and Parkinson's diseases. It should be emphasized, however, that the molecules that are the subject of the following mechanistic studies also have antioxidant characteristics. This is especially the case for the polyphenol EGCG, which is a potent antioxidant that can also complex iron and copper ions and thus neutralize their catalytic effects in the production of reactive oxygen species (ROS). The mechanisms involved in this activity have been described extensively elsewhere (Jomova, Vondrakova et al.; Mandel, Amit et al. 2008; Zhao 2009).
Within the framework of the amyloid hypothesis, it seems that the oligomeric intermediate stages of the amyloid cascade are the main culprits for the toxicity of misfolded proteins (Walsh, Klyubin et al. 2002; Shankar, Li et al. 2008). Therefore, various strategies can be applied to reduce the amount of these toxic intermediates (Fig. 1). The first strategy is directed toward cutting off the supply of aggregation-competent monomeric protein; an example of this is the use of β-Sekretase inhibitors (Haass, Koo et al. 1992; Vassar 2001) that block the formation of the Aβ peptide via cleavage of the amyloid precursor protein (APP). A second approach is the inhibition of aggregation with substances that bind to and destabilize the amyloid structure (Fig. 1). See Cohen and Kelly (2003) for a detailed review.
In the present study a novel therapeutic strategy will be examined: the redirection of the amyloid cascade towards nontoxic aggregates and the stabilization of larger amyloid fibrils (Fig. 1). In both cases, the concentration of aggregation intermediates is reduced: in the first case the irreversible formation of off-pathway aggregates is favoured, and in the second the formation of stable fibrillar structures is promoted.
New strategies for intervention in amyloidogenesis: EGCG
Catechins from tea leaves and grape seeds, especially (-) epigallocatechin gallate (EGCG), have been a focus of intensive research for more than a decade. Tea leaves consist of 30% catechins; one third of this is EGCG (Graham 1992). In addition to its antioxidant properties, EGCG shows promising effects on tumour growth in cell and mouse models. Thus, a possible application in cancer therapy has been extensively investigated. The various areas of potential EGCG applications have been summarized by Zaveri (2006).
EGCG reduces the toxicity of Aβ in pheochromocytoma and neuroblastoma cell models (Choi, Jung et al. 2001; Li, Jang et al. 2004). EGCG decreases the formation of amyloid deposits (Rezai-Zadeh, Shytle et al. 2005; Rezai-Zadeh, Arendash et al. 2008) and the loss of spatial learning ability in a mouse model of Alzheimer's disease (Wang, Ho et al. 2008). In a rat model, there was an increase in learning ability with long-term EGCG treatment (Haque, Hashimoto et al. 2008). EGCG or green tea extract inhibit the loss of dopaminergic neurons in the substantia nigra in animal models of Parkinson's disease (Levites, Weinreb et al. 2001). Epidemiological studies in which patients were asked over many years about their lifestyles also showed a negative correlation between the consumption of green tea and loss of cognitive function (Kuriyama, Hozawa et al. 2006). The incidence of Parkinsonism is 5- to 10-fold lower in Asia than in Western societies (Zhang and Roman 1993). Finally, the first clinical pilot studies demonstrated a reduction in LC amyloid deposition and the thickness of the cardiac septum in myeloma patients after the intake of green tea extract (Mereles, Buss et al. 2010; Hunstein 2007).
Mechanism of the protective effect of EGCG
The mechanism by which EGCG reduces the toxicity of amyloid remained unknown for a long time. Early hypotheses centred on the antioxidant activity of EGCG (Mandel, Amit et al. 2008, see Section 1.5). It has been described, however, that EGCG increases the degradation of APP into non-amyloidogenic peptides via α-secretase (Fernandez, Rezai-Zadeh et al. 2010; Lee, Lee et al. 2009) and inhibits the activity of -secretase (Jeon, Bae et al. 2003).
In 2006, Dagmar Ehrnhoefer demonstrated for the first time that EGCG directly inhibits the aggregation of a misfolded protein (Ehrnhoefer, Duennwald et al. 2006). In the meantime it has been shown for many amyloidogenic proteins that EGCG binds directly to the protein and inhibits the formation of amyloid fibrils, including α-synuclein (αS, Bae, Kim et al. 2010; Ehrnhoefer, Bieschke et al. 2008), Aβ (Ehrnhoefer, Bieschke et al. 2008; Ono, Condron et al. 2008), transthyretin (Ferreira, Cardoso et al. 2009), lysozyme (He, Zhao et al. 2009), the prion protein PrP (Rambold, Miesbauer et al. 2008), and the yeast prion Sup35 (Roberts, Duennwald et al. 2009).
EGCG binds in particular to unfolded proteins (Ehrnhoefer, Bieschke et al. 2008; Wang, Liu et al. 2010). Examination by NMR of the binding of EGCG to αS showed an extinction of the resonance of 30% of the amino acids (Ehrnhoefer, Bieschke et al. 2008). This points to a non-specific interaction with hydrophobic moieties where the binding could occur through hydrophobic interactions or through H-bonding. EGCG binds to the Aβ peptide via hydrophobic sequences at positions 14-24 and 27-37 (M. Lopez, B. Reif, personal communication; Grelle, Albrecht et al. 2011). This inhibits the formation of the β-sheet in the region around positions 14-24, which is thought to initiate the formation of amyloid (Kim and Hecht 2006). EGCG can also bind to the hydrophobic region of folded proteins such as albumin (Maiti, Ghosh et al. 2006) or transthyretin (Miyata, Sato et al. 2010). Several defined binding sites were localized on exposed hydrophobic residues of the proteins. The binding energy has an entropic as well as an enthalpic component, which indicates that stabilization of H-bonds occurs in addition to the hydrophobic interactions (Maiti, Ghosh et al. 2006). A thermodynamic analysis of the interaction of EGCG with Aβ40 confirmed that there are entropic and enthalpic contributions to the binding (Wang, Liu et al. 2010). A reversible covalent conjugation, e.g. via the formation of Schiff bases, has also been discussed (Ishii, Ichikawa et al. 2011).
The analysis of aggregation reactions in the presence and absence of EGCG demonstrated that EGCG inhibits the formation of fibrillar aggregates that bind to amyloid dyes such as Congo red or thioflavin T. Instead, in the case of αS, Aβ and most other amyloidogenic proteins, stable, spherical aggregates are formed (Fig. 2, Ehrnhoefer, Bieschke et al. 2008). These spherical aggregates are not cytotoxic and have a small proportion of β-sheet existing as fibrils or amyloid intermediates. In particular, they lack the characteristic property of amyloid proteins to catalyse their own formation. If they are added to a solution of monomeric Aβ or αS in the presence of EGCG, there is no acceleration of the aggregation kinetics. The EGCG aggregates cannot function as aggregation nuclei because they are off-pathway in relation to the amyloid cascade (Fig. 2).
This mechanism has now been confirmed in various protein systems, e.g. for different forms of αS (Bae, Kim et al. 2010), lysozyme (He, Xing et al. 2009), transthyretin (Ferreira, Saraiva et al. 2011) and the yeast prion Sup35 (Roberts, Duennwald et al. 2009). However, it is not fully universal but rather depends on the stability of the protein aggregates formed. Following carboxymethylation casein forms fibrils with β-sheet structure that bind to the amyloid dye thioflavin T; it is thus used as a generic model system for amyloid formation (Ecroyd, Koudelka et al. 2008). EGCG inhibits the fibril formation of casein through its binding to a β-sheet-turn-sheet motif of the protein (Hudson, Ecroyd et al. 2009). The protein remains in solution as a monomer after EGCG binding. The casein model, however, is atypical for amyloidogenic proteins in that the β-sheet-turn-sheet motif structure, which is characteristic of many amyloid fibrils (Luhrs, Ritter et al. 2005), is already present in the monomeric form (Hudson, Ecroyd et al. 2009). In contrast, for the Aβ peptide as well as most other proteins, these types of structures are present in the oligomeric and protofibrillar intermediates of the amyloid cascade (Mastrangelo, Ahmed et al. 2006) and are not found in the monomeric protein.
Disassembly and remodelling of amyloid deposits
Hauber, Hohenberg et al. (2009) demonstrated that EGCG inhibits the formation of amyloid-type fibrils of a fragment of prostatic acidic phosphatase (PAP) and dissolves fibrils that have already formed. The amyloid fibrils of this semen-derived enhancer of virus infection (SEVI) bind to human immunodeficiency virus (HIV) and facilitate its entry into the body through mucous membranes (Münch, Rücker et al. 2007); here, a local application of EGCG helps to prevent HIV infection (Hauber, Hohenberg et al. 2009). Disassembly or transformation of fibrils by EGCG has also been observed for several other amyloid-forming proteins. After treatment with EGCG, the yeast prion Sup35 loses its ability to replicate the fibrillar structure (Roberts, Duennwald et al. 2009). EGCG degrades fibrils of transthyretin (Ferreira, Saraiva et al. 2011); fibrils of Merozoite surface protein 2 (MSP2) are also transformed into an amorphous structure by EGCG (Chandrashekaran, Adda et al. 2011). We were able to clarify the mechanism for the first time for αS and Aβ fibrils (Bieschke, Russ et al. 2010). When EGCG is added to amyloid fibrils of Aβ or αS that have already formed, the fibrillar structure is gradually lost and SDS-resistant spherical or amorphous structures are formed that do not bind to thioflavin T, do not catalyse seeding-induced aggregation, and are not toxic in neuronal (SHSY5Y, PC12) cell models (Bieschke, Russ et al. 2010). They possess the same properties as the off-pathway aggregates that are formed in the presence of monomeric αS and Aβ in the presence of EGCG. In the neuronal cell model Aβ aggregates are degraded after treatment with EGCG (Fig. 3).
Mechanisms of amyloid fibril remodelling
Two mechanisms may be postulated for the transformation of fibrillar into amorphous structures: either the fibrils are dissociated by EGCG or they are converted directly into amorphous structures, i.e. without passing through the monomer stage (Fig. 4Ai). Direct disassembly of the fibrils would be possible if EGCG binds to them, or it could occur indirectly if EGCG binds to the monomeric form and shifts the equilibrium between monomeric and fibrillar phases by removing monomers irreversibly from the equation (Fig. 4Aii).
In order to differentiate between these two possibilities, fibrils were produced with built-in fluorescent markers. If red-tagged fibrils are mixed with green-tagged fibrils, then disassembly of the fibrils followed by re-aggregation should lead to amorphous aggregates containing the same amount of red- and green-tagged monomer (which would be yellow). If the fibrils are transformed by EGCG in situ into amorphous structures without first being disassembled, then each structure would either be predominantly green or red (Fig. 4B). Surprisingly, this latter scenario was observed after treating Aβ with EGCG (Bieschke, Russ et al. 2010), indicating that EGCG does not disassemble the fibrils but rather remodels them directly into amorphous structures. The molecular details of this mechanism still need to be clarified. It is conceivable that EGCG binds to the β-sheet of Aβ in the fibrils, such that the cross-β structure is weakened, while it simultaneously promotes aggregation through hydrophobic interactions, Π-Π stacking, or the formation of covalent networks.
Perspectives for prevention and therapy
A widely recognized problem in the therapeutic application of EGCG is the large variability in the bioavailability of orally administered EGCG (reviewed in Singh, Shankar et al. 2011; Mereles and Hunstein 2011). This is due to the high sensitivity to oxidation, the tendency to conjugate to proteins in the digestive tract (e.g. casein), and the rapid metabolism in the body. With an especially conscientious oral regimen these problems can be overcome (Hunstein 2007). However, the variations in bioavailability confound the systematic evaluation of treatment outcome in clinical studies (Mereles and Hunstein 2011). In addition, only 10-20% of the EGCG passes the blood-brain barrier (Singh, Arseneault et al. 2008). There are now various approaches to solving the problem of bioavailability: EGCG in capsule form increases the uptake and the long-term bioavailability (Dube, Nicolazzo et al. 2010; Siddiqui, Adhami et al. 2010). The uptake of EGCG in the gastrointestinal tract can be increased via the simultaneous intake of vitamin C (Peters, Green et al. 2010) and piperine (Lambert, Hong et al. 2004). Concomitant intake of fish oil augmented the anti-amyloid effectiveness of EGCG in a mouse model of Alzheimer's disease (Giunta, Hou et al. 2010).
There are also ways of improving the cellular availability of EGCG. Enclosing EGCG in poly-(lactide-co-glycolide) nanoparticles increases the enhancing effect of EGCG in chemotherapy with cisplatin compounds (Singh, Bhatnagar et al. 2011). Enclosure in lipid nanoparticles increased the effect of EGCG on the cleavage of APP by α-secretase in neuroblastoma cells (Smith, Giunta et al. 2010). The transport of small molecules like EGCG through the cell membrane can also be augmented through irradiation with a pulsed laser source in the red to NIR wavelength region (Sommer, Zhu et al. 2010). NIR laser irradiation penetrates deeply into human tissue and can stimulate mitochondrial metabolism (Eells, Henry et al. 2003) and enhance the effectiveness of EGCG in preventing the proliferation of tumour cells (Sommer, Zhu et al. 2010). The combination of pulsed laser irradiation at 670 nm with the intake of EGCG decreases the amount of Aβ aggregates and improves the metabolic activity in a neuroblastoma cell model (Sommer, Bieschke et al. 2011).
The misfolding of endogenous proteins and the formation of fibrillar structures, which are integral to the concept of amyloidogenesis, is a complex process with many intermediate steps. The complicated mechanism hinders a complete understanding of amyloid formation and toxicity but provides many potential targets that can be addressed therapeutically. Oligomeric intermediates play a central role in the pathology of disorders of protein misfolding. They are the main source of toxicity. The intervention described here with EGCG targets precisely this point in the amyloid cascade. Before successful therapeutic application of the polyphenol EGCG in protein misfolding disorders in human patients, however, there are still numerous practical hurdles that have to be overcome.
Nevertheless, clarification of the basic mechanisms is the foundation on which every rational therapy is built. It offers a starting point for improving the pharmacokinetic properties or for the development of new substances that have the same mechanism of action as the tea polyphenols described here. The present investigations offer new treatment strategies for amyloid disorders and show as a proof-of-principle that detoxification of amyloid structures and redirection of the fibril formation process is possible. It is my express hope that the results described here will contribute to the discovery of an effective, cause-based therapy for Alzheimer's and Parkinson's diseases and other destructive forms of protein misfolding.
The present text is a selection of excerpts from the Habilitation thesis of Dr. Jan Bierschke. All rights remain with the Author.
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Appendix: Additional figures from Ilona Hauber, Heinrich Hohenberg, Barbara Holstermann, Werner Hunstein and Joachim Hauber. PNAS 2009;106:9033-9038.
Figures: Heinrich Hohenberg and Rudolph Reimer (Heinrich-Pette-Institut, Hamburg).
Analysis of amyloid fribrils of human semen using transmission electron microscopy (TEM); 17,000-times magnified. One part (peptide) of a prostate enzyme of prostatic acid phosphatase (PAP) forms amyloid fibrils in human semen. These significantly enhance infectiousness of HIV-1 under laboratory conditions. It is therefore probable that they support the sexual transmission of HIV-1.
SEVI: semen-derived enhancer of virus infection. Transmission electron microscopy analysis of EGCG-treated SEVI in a closed system. Corresponding images are arranged side by side. (A) Dissolved EGCG is shown inside the ultrathin sectioned microtubes and at different magnifications. The overview shows the interface between the tube wall (arrow) and the tube's lumen filled with EGCG (magnification: 3000×). EGCG aggregates are organized in different patterns (magnifications: 35,000× and 75,000×). (B) The SEVI solution shows a high density of SEVI-specific amyloid fibrils (magnification: 3,000×). Because of the embedding angle, the fibrils often run out of the section plane (magnification: 28,000×). Nevertheless, their length differs between 30 and 90 nm. (C) Appearance of SEVI after 12 h of incubation in EGCG. The small EGCG aggregates diffused from the outside through the porous membrane into the lumen of the tube covering the surface of the fibrils (magnification: 6,300×). (D) Degradation of the majority of SEVI after 60 h of incubation in 10 mM EGCG.
Figurel A: Intact fibrils have a characteristical filamentous structure.
Figure B: After 60 hrs of incubation in EGCG an ingredient of green tea, fibrils have dissolved almost completely.