In vitro studies: an update

In vitro studies on the anti-neuroinflammatory effects of EGCG have been performed on different cells lines including MC65, EOC 13.31, SweAPP N2a, N2a/APP695, DIV8, CHO, and M146 L cells (Table 1). Results from these studies showed that the anti-neuroinflammatory capacity of EGCG is mainly associated to the inhibition of microglia-induced cytotoxicity.

Lin et al. demonstrated that EGCG was able to suppress the neurotoxicity induced by Aβ, through the activation of the glycogen synthase kinase-3β (GSK-3β) and the inhibition of c-Abl/FE65 nuclear translocation [20]. This was a relevant finding, since it is of note that c-Abl is a cytoplasmic nonreceptor tyrosine kinase involved in the development of the nervous system and implicated in the regulation of cell apoptosis, whereas the β-isoform of GSK3 is a proline-directed serine-threonine kinase involved in neuronal cell development and energy metabolism [21]. These data suggest that c-Abl/GSK-3β signaling is involved in neuronal loss, neuroinflammation and gliosis. It is of note that, related to AD’s pathogenesis, the proteolytic processing of a transmembrane glycoprotein, known as amyloid precursor protein (APP), is responsible for the Aβ’s origin [22]. Other investigations have been conducted to evaluate the effect of EGCG on Aβ-induced inflammatory responses in microglia. To this regard, Wei et al., investigated on the inhibitory effects of EGCG on microglial activation induced by Aβ and on neurotoxicity in Aβ-stimulated EOC 13.31 microglia. Results revealed that that EGCG was able to suppress the expression of TNFα, IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) and to restore the levels of intracellular antioxidants against free radical-induced pro-inflammatory effects in microglia, the nuclear erythroid-2 related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) [23]. In addition, EGCG suppressed Aβ-induced cytotoxicity by reducing ROS-induced NF-κB activation and mitogen-activated protein kinase (MAPK) signaling, including c-Jun N-terminal kinase (JNK) and p38 signaling. Taken together these data suggest that EGCG is able to inhibit the neuroinflammatory response of microglia induced by Aβ and to protect against indirect neurotoxicity through several mechanisms.

Previously, Rezai-Zadeh et al. demonstrated that GTE reduced the generation of Aβ in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant APP (SweAPP N2a cells) and activated the nonamyloidogenic processing of APP, also promoting its α-secretase cleavage [24].

One of the processes involved in the amyloid formation cascade, is the β-sheet formation. This event is frequently associated with cellular toxicity in many of human protein misfolding diseases, including AD. It has been reported that EGCG was able to interfere with this cascade, by redirection of prone polypeptides’ aggregation into off-pathway protein assemblies [25]. In another study, Bieschke et al. showed that EGCG converted the large mature Aβ fibrils into smaller forms with no toxicity for mammalian cell [26]. A recent study conducted by Chesser et al., proved that EGCG in DIV8 primary rat cortical neurons, was able to enhance the clearance of AD-relevant phosphorylated tau species, indicating that EGCG could be used as an adjuvant agent for AD’s treatment [27]. Similar findings were obtained by Chang et al. The authors demonstrated that EGCG reduced β-amyloid (Aβ) accumulation in M146 L and CHO cells [28]. Finally, very recently it has been reported that EGCG inhibited β-Amyloid generation and oxidative stress involvement of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in N2a/APP695 cells [29].

All these data suggest that EGCG may be considered an important agent with neuroprotective properties against AD.

In vivo preclinical studies: an-update

The neuroprotective effects of EGCG have been also demonstrated by in vivo experiments on several animal models (Table 2). The first study was reported by Rezai-Zadeh et al. [24]. The authors described that EGCG was able to decrease Aβ levels and plaques formation in a transgenic mouse model of “Swedish” mutant APP when was injected intraperitoneally (20 mg/kg). Similar results were obtained by the same group of researchers, when EGCG administered orally in drinking water (50 mg/kg), reduced Aβ deposition in the same mutant mice [30]. In another study based on the generation of transgenic mouse models of AD, Li et al., [31] investigated on EGCG (orally 20 mg/kg/day, for 3 months) capacity to interfere with Aβ deposits in different brain areas. Data emerged by immunohistochemistry, showed that Aβ deposits were reduced by 60% in the frontal cortex and 52% in the hippocampus. A reduction of Thioflavine-S histochemistry labelling compact plaques was also detected in both regions. In addition, the percentage of CD45, a marker of microglial activation, was lower than 18% in the cortex and then 28% in the hippocampus respect to those observed in the control cohort. New insights on the role of EGCG in AD’s treatment have been reported by Smith et al. [32]. The authors engineered nanolipidic EGCG particles to improve oral’s bioavailability of EGCG. By using this system in mouse model of AD’s disease, the ability of EGCG for the treatment of AD was enhanced more than two-fold respect to treatment with free EGCG.

The role of EGCG in AD’s treatment, was also described by Giunta et al., in an interesting study in which was used Fish oil (8 mg/kg/day) combined to EGCG (62.5 mg/kg/day or 12.5 mg/kg/day) in a mouse model of AD. Results obtained from these experiments, showed that co-treatment of N2a cells with fish oil and EGCG increased the production of sAPP-alpha respect to either compound alone, indicating that these compounds were able to make a synergetic action on the inhibition of cerebral Aβ deposits [33].

More recently, Lee et al. [19] tested the effects of EGCG on neuroinflammation and amyloidogenesis, in mice with systemic inflammation. The authors demonstrated that EGCG was able to prevent memory impairment induced by lipopolysaccharide (LPS) and apoptotic neuronal cell death. Moreover, EGCG prevented LPS-induced activation of astrocytes and increased cytokines expression (TNF-α, IL-1β, IL-6), suggesting that EGCG could be considered a therapeutic agent for neuroinflammation-associated AD. In animal another model of dementia, generated by infusion of streptozotocin (STZ) into intracerebroventricular (ICV) of rats, Biasibetti et al., showed that EGCG (10 mg/kg/day for 4 weeks) completely abrogated the cognitive deficit by influencing the glial-specific calcium binding protein S100B content in the hippocampus, the acetylcholinesterase (AChE) activity, the glutathione peroxidase activity, the nitric oxide (NO) metabolites, and ROS content [34].

He et al., by using an AD’s mouse model generated by D-gal, showed that EGCG had a protective effect on AD by decreasing the expression of APP and Aβ in the hippocampus of mice [35]. Similarly, Lin et al. proved that EGCG impaired the formation of Aβ, through the inhibition of APP proteolysis, cAbl/FE65 complex nuclear translocation and GSK3 activation [20].

Since insulin signaling plays a significant role in the regulation of synaptic activities involved in learning and memory processes, insulin resistance – expressed as increased phosphorylation levels of insulin receptor substrate-1 (IRS-1) - and subsequent alteration in signaling pathways, may contribute to the cognition impairment in AD’s patients [36]. Interestingly, Jia et al. demonstrated that EGCG reduced the spatial memory impairment (AD-related cognitive deficit) in APP/PS1 mice (bearing brain insulin resistance) by inducing IRS-1 signaling defects in the hippocampus, in a dose dependent manner [37].

In another study, was reported that EGCG reduced Aβ accumulation in vitro and rescued cognitive deterioration in senescence-accelerated mice P8 (SAMP8). The authors showed that EGCG attenuated the cognitive deterioration in AD’s mouse model by upregulation of neprilysin (NEP) expression [28].

Taken together, these data strongly suggest that EGCG could be used as a therapeutic agent for the treatment and the prevention of AD.

The bioavailability of EGCG in the brain: a discrepancy between animals and humans-based studies

Despite these encouraging results, there is still a translational gap between in vitro, in vivo and clinical studies with EGCG in neurodegenetative disease treatment. This can be associated to poor data reported on the bioavailability of EGCG in the brain, a feature extremely necessary for its neuroprotrective role. In vivo studies performed on animal models showed that repeated administration of EGCG, increased its accumulation in the brain [41, 42]. Opposite results were obtained in a study performed on six human subjects which assumed green tea by drinking. The products of green tea’s metabolism, (i.e. flavan-3-ol methyl-glucuronide and sulfate metabolites) were not able to reach the brain, thus remaining in the bloodstream [43]. These discrepancy can be associated to different causes, as reported by Mähler et al.in a review on this topic (i.e. dose, time point of EGCG treatment and different catechin metabolism between animals and humans) [39]. When EGCG is able to reach the brain, it regulates many biological processes and molecular signaling pathways involved in neurodegenerative disorders, included AD, as previously reported based on convincing results of in vitro and in vivo pre-clinical studies [44]. Unfortunately, these mechanisms are not completely elucidated in clinical studies probably due to the absence of standardization of disease severity in humans and in green tea preparations and its derivatives.

This strongly suggest the needing of more detailed and specific studies on EGCG’s brain and plasma bioavailability, on its efficacy and safety in patients, and on possible interactions with other drugs.

Clinical trials

Despite the encouraging data obtained from pre-clinical studies, several pivotal issues, regarding EGCG dose levels and administration frequency as well as genetic and epigenetic modulations involved in the metabolism and distribution of the active compounds in humans, remain to be explored [45].Previous findings from a cross-sectional study showed a negative association between green tea consumption and the prevalence of cognitive impairment in elderly individuals over 70 years old [46]. Thus, there is a discrepancy about the effects of GTE compounds on cognitive functions [47].

Several clinical studies have been performed to evaluate the acute effects of EGCG and other constituents of tea, such as L-theanine, on cognitive function (e.g., attention) and mood. The results obtained, showed that tea consumption had significant acute benefits on mood and work performance and creativity [48, 49]. Another clinical study performed on 27 healthy human adults treated with EGCG (orally administered in a single dose of 135 mg), reported that EGCG was able to modulate cerebral blood flow parameters, without affecting cognitive performance or mood [50]. Similarly, Scholey et al. showed that EGCG administration (300 mg) was associated with reduced stress, increased calmness and increased electroencephalographic activity (increased alpha, beta and theta activities) in the midline frontal and central brain regions [51]. Ide et al. showed that green tea consumption in subjects with cognitive dysfunction (2 g/day for 3 months, approximately equal to 2 to 4 cups of tea/day) significantly improved cognitive performance [52].

It is of note that the clinical symptoms of AD do not occur immediately. For this reasons, acute results of EGCG or other natural compounds on neurocognitive capacities, cannot be predictive of efficacy in more complex neurodegenerative diseases, such as AD’s syndrome. To date, one ongoing clinical trial is investigating on the effects of EGEG in early state of AD patients co-medicated with acetylcholine esterase inhibitors (ClinicalTrials.gov identifier: NCT00951834) [53].

In order to evaluate the clinical effects of EGCG on AD, are necessary: (i) more detailed in vitro and in vitro studies with the purpose of dissect the underlying molecular mechanisms by which EGCG interferes with AD pathogenesis; (ii) clinical studies exploring the long-term effects of EGCG on cognitive functions; (iii) large size epidemiological studies concerning the consumption of EGCG and the progression of AD.