Abstract
Background:
Green tea has been widely recognized in ameliorating cognitive impairment and Alzheimer’s disease (AD), especially the progression of cognitive dysfunction. But the underlying mechanism is still unclear.
Objective:
This study was designed to determine the role of green tea consumption in the association with cerebrospinal fluid (CSF) biomarkers of AD pathology and to ascertain whether specific population backgrounds showed the differences toward these relationships.
Methods:
Multivariate linear models analyzed the available data on CSF biomarkers and frequency of green tea consumption of 722 cognitively intact participants from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE) database, and we additionally detected the interaction effects of tea consumption with APOE ɛ4 status and gender using a two-way analysis of covariance.
Results:
Frequent green tea consumption was associated with a decreased level of CSF total-tau protein (t-tau) (p = 0.041) but not with the levels of CSF amyloid-β 42 (Aβ42) and CSF phosphorylated tau. The more pronounced associations of green tea consumption with CSF t-tau (p = 0.007) and CSF t-tau/Aβ42 (p = 0.039) were observed in individuals aged 65 years or younger. Additionally, males with frequent green tea consumption had a significantly low level of CSF t-tau/Aβ42 and a modest trend toward decreased CSF t-tau. There were no interaction effects of green tea consumption with APOE ɛ4 and gender.
Conclusion:
Collectively, our findings consolidated the favorable effects of green tea on the mitigation of AD risk. The constituents of green tea may improve abnormal tau metabolism and are promising targets in interventions and drug therapies.
INTRODUCTION
Alzheimer’s disease (AD) has been a gradually growing public health concern, as a result of incremental prevalence and mortality rates, as well as heavy burden on caregivers and society [1]. Although well-established research has uncovered a great deal about AD, much is yet to be focused on about how to prevent or slow the disease, and how the genetic, environmental, and lifestyle factors are correlated to AD biological changes. Like chronic diseases, AD is deemed to be a neurodegenerative disease driven by multi-factors rather than a single cause [1]. Of note, epidemiological research provides substantial evidence that lifestyle factors as modifiable manners can reduce an individual’s risk of dementia in later life or delay the occurrence of AD [2–4].
Tea, particularly green tea with abundant catechins mostly including (–)-epigallocatechin 3-gallate (EGCG) and L-theanine, has been corroborated to have a favorable effect on low incidence of chronic pathologies [5]. Both epidemiological [6] and systematic studies [7, 8] have shown green tea consumption as one modifiable lifestyle factor that can help to ameliorate cognitive impairment and AD, especially the progression of cognitive dysfunction [9]. Mechanistic studies revealed that green tea catechins invoked extensive cellular mechanisms related to antioxidant, as well as potent activities of neuroprotection and neurorescue, which might ameliorate cognitive dysfunction in AD and thereby halting the progression of cognitive decline [5, 10–12]. Indeed, intake of green tea (which contains high levels of catechins) can facilitate preserving attention and memory [10, 13] and produce a better neuroprotective effect than black tea [14]. Animal studies also revealed that long-term oral consumption of EGCG ameliorated impairments in spatial memory and rescued the abnormal synaptic protein levels in AD mice models [15, 16]. As such, it is plausible to hypothesize that green tea consumption may mitigate the main pathological changes of AD. However, few relevant population-based studies investigated the exact mechanism underlying the associations between tea consumption and the pathogenesis of AD.
The alterations of cerebrospinal fluid (CSF) core biomarkers of AD have been deemed to reflect underlying pathologic damage of the brain, which is essential to identify individuals in preclinical and mild cognitive impairment (MCI) stages of AD [17–20]. Thus, the primary objective of our investigation was to determine whether green tea consumption was related to biological changes in AD pathology. This is the first exploration to assess the association between green tea consumption and CSF biomarkers of AD in cognitively intact older adults from a large-scale study in the Han Chinese population, with the purpose of providing novel clues for fundamental research and longitudinal studies about the prevention and drug development.
METHODS
CABLE study
All participants included in our study were recruited from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE) study. Since 2017, CABLE is an ongoing large-scale cohort study and aims to identify genetic, environmental, and lifestyle risk factors and biomarkers of AD in Chinese Han population. All participants in CABLE were enrolled at Qingdao Municipal Hospital, Shandong Province, China. The exclusion criteria included: 1) central nervous system infection, head trauma, epilepsy, multiple sclerosis, or other major neurological disorders; 2) major psychological disorders (e.g., depression); 3) severe systemic diseases (e.g., malignant tumors); 4) family history of genetic disease. All participants underwent clinical and neuropsychological assessment, biochemical testing, as well as blood and CSF sample collection. Demographic information, AD risk factor profile, and medical history were also collected by comprehensive questionnaires and an electronic medical record system [21]. Participants were aged between 40 to 90 years and consisted of cognitively intact older adults and individuals with MCI or AD. Each participant received a consensus diagnosis by the professional medical doctors through intact performance on neuropsychological testing, combined with CSF biomarkers and brain magnetic resonance imaging (MRI) examinations in compliance with the National Institute on Aging–Alzheimer’s Association (NIA-AA) workgroup diagnostic criteria [22, 23].
The CABLE study was in accordance with the Helsinki declaration, and the protocol for this study was approved by the Institutional Ethics Committees of Qingdao Municipal Hospital. All enrolled participants or their caregivers signed written informed consents.
Study participants
This study included 722 cognitively intact participants from the CABLE study who failed to meet the criteria of MCI and AD. An adapted Chinese-Modified Mini-Mental State Examination (CM-MMSE) and a Montreal Cognitive Assessment (MoCA) were used to assess the general cognitive function. The tests were administered by specially trained neurologists. The CM-MMSE is a fully structured screening instrument evaluating questions about memory, orientation to time and place, registration, attention and calculation, recall, language, and visual construction. Possible scores of this scale range from 0 to 30 with higher scores representing better cognitive performance. Relevant information of each participant was obtained from the CABLE cohort, including age, gender, years of education, CM-MMSE, Apolipoprotein E ɛ4 (APOE ɛ4) status, and levels of CSF biomarkers of AD pathology (amyloid-β 42 [Aβ42], total-tau protein [t-tau], phosphorylated tau [p-tau], t-tau/Aβ42, and p-tau/Aβ42), as well as details about green tea consumptions.
Green tea consumption
For each participant, green tea consumption data was collected by trained research staff using a validated self-reported questionnaire. Two items were adopted in the questionnaire about whether drinking green tea regularly and the frequency of green tea consumption. The options of first item were recorded as yes or no. The green tea consumption was further quantified by the frequency of consumption using the choice of never, <1, 1–2, 3, 4–6 times/week, or almost every day. To increase sample sizes of diverse frequency groups, as well as compare the effects of tea consumption frequency on the CSF biomarkers, individuals who drank tea less than three times a week were grouped as the reference group (infrequent green tea consumption group), and the others were assigned into the frequent tea consumption group, which was consistent with previously published studies [24–26].
Measurements of CSF AD biomarkers
The CSF specimens were collected from participants by lumbar puncture in the morning after overnight fasting. These specimens were centrifuged at 2000×g for 10 min to eliminate cells and other insoluble materials within 2 h after collection, and were snap frozen at – 80°C until assay. CSF Aβ42, t-tau, and p-tau were detected by the enzyme-linked immunosorbent assay (ELISA) kit (Innotest β-AMYLOID (1–42), PHOSPHO-TAU (181p), and hTAU-Ag; Fujirebio, Ghent, Belgium) on the microplate reader (Thermo Scientific™ Multiskan™ MK3). The standards and CSF specimens were analyzed in duplicates, and the means values of the duplicates were used for subsequent statistical analyses. The mean inter-assay coefficient of variation was under 15% (8.79% for Aβ42, 11.34% for p-tau, and 10.38% for t-tau). The mean intra-assay coefficient of variation was under 10% (5.00% for Aβ42, 2.33% for p-tau, and 4.70% for t-tau). All analyses were operated by professional experimenters who were blind to clinical information.
APOE genotyping
Fasting blood specimens were centrifuged at 2000×g for 10 min after collection and stored at – 20°C until assay. The thaw-freezing cycle was limited not to surpass two times. DNA was extracted from these specimens using QIAamp®DNA Blood Mini Kit (250) and stored in an enzyme-free EP tube at – 80°C until the APOE ɛ4 genotyping. Restriction fragment length polymorphism (RFLP) technology was applied for genotyping according to two specific loci, including rs7412 and rs429358. APOE ɛ4 carrier status was classified into APOE ɛ4 non-carriers or carriers with at least one ɛ4 allele.
Statistical analysis
All statistical analyses were performed using R (version 3.6.2) and SPSS (version 25.0, IBM) software programs. A two-tailed p < 0.05 was considered statistically significant. The data were shown in the form of mean±SD (standard deviation) or proportions of them. The outlier values which situated outside three SD were excluded prior to subsequent analyses. Group differences in demographic characteristics and levels of CSF core biomarkers were tested using the chi-square analyses for categorical variables, as well as the independent samples t-test or Mann-Whitney U test for continuous variables. In terms of skewed data (Shapiro-Wilk test <0.05), the Box-Cox transformations were performed to construct these data with approximately normal distributions via “car” package of R software.
To determine the associations between green tea consumption (frequent or infrequent) and CSF biomarkers of Alzheimer’s pathology (Aβ42, t-tau, p-tau, t-tau/Aβ42, and p-tau/Aβ42), the multivariate linear regression analyses were performed with CSF biomarkers as dependent variables and tea consumption as independent variable. Each linear model was adjusted for age, gender (male or female), years of education, APOE ɛ4 status (carriers or non-carriers), and CM-MMSE. The multicollinearity was assessed using tolerance, Variance Inflation Factor (VIF), and Pearson’s correlation coefficients. No multicollinearity existed in each model of current study. Further subgroup analyses stratified by age (age >65 years and age ≤65 years) and gender were conducted to explore that whether potential associations were driven by specific demographic characteristics. To determine the effects of green tea consumption on CSF biomarkers independent of APOE ɛ4 status and gender as modifiers, a two-way analysis of covariance (ANCOVA) was performed to assess the interaction effect of tea consumption with APOE ɛ4 status or gender. Main effects and interaction terms were both included in this analysis.
RESULTS
Demographic characteristics of participants
Table 1 showed the demographic characteristics of participants between frequent and infrequent green tea consumption groups. A total of 722 individuals who underwent measurements of CSF biomarkers and questionnaires of green tea consumption were eventually included. Overall, the included population were cognitively intact (mean CM-MMSE score = 27.77) with a 59.70% proportion of males, and the mean age was 62.41 years (SD = 10.39 years). The APOE ɛ4 carriers accounted for roughly 15.70%. There were no significant differences in age, years of education, CM-MMSE score, and APOE ɛ4 status between frequent and infrequent green tea consumption groups. Of note, the proportion of males in frequent group was statistically higher in comparison with the proportion in infrequent group (p < 0.001).
Characteristics of included participants from CABLE database
Date are reported as mean (SD). p values of between-group comparisons were obtained using the t-test, Mann-Whitney U test or chi-square test. CM-MMSE, China-Modified Mini Mental State Examination; APOE ɛ4, apolipoprotein epsilon-ɛ4; CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau; p-tau, phosphorylated tau; SD, standardized deviation.
Green tea consumption and CSF biomarkers of AD pathology in total participants
In terms of between-group differences of individual CSF biomarkers and combinations of them, significantly lower levels of CSF t-tau (p < 0.018) and CSF t-tau/Aβ42 (p < 0.013) were observed in frequent tea consumption group when compared to those in infrequent group (Table 1). There were no differences in levels of CSF Aβ42, CSF p-tau, and CSF p-tau/Aβ42 between two green tea consumption groups (p > 0.05).
Table 2 summarized the linear regression results of associations between green tea consumption and CSF biomarkers. Frequent tea consumption was significantly associated with high level of CSF t-tau (β= –0.00428, p = 0.041), indicating a negative link between tea consumption and abnormal tau pathology. We failed to find the associations of tea consumption with CSF Aβ42, CSF p-tau, CSF p-tau/Aβ42, as well as CSF t-tau/Aβ42 (all p > 0.05).
Correlations between green tea consumption and CSF biomarkers of Alzheimer’s disease pathology
The β coefficients and p values were calculated using multiple linear regression models after adjustment for age, gender, year of education, APOE ɛ4 status, CM-MMSE. The Box-Cox transformations were applied for the normalization of all data of CSF biomarkers as dependent variables. CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau; p-tau, phosphorylated tau.
Stratified analyses of associations between green tea consumption and CSF biomarkers
To explore whether significant associations were driven by specific demographic characteristics, we next conducted subgroup analyses stratified by age and gender. Of note, there were negative and significant associations of green tea consumption with CSF t-tau (β= –0.00744, p = 0.007) (Fig. 1A) and CSF t-tau/Aβ42 (β= –0.08232, p = 0.039) (Fig. 1B) in individuals aged 65 years or younger. But no similar correlations were found in those aged older than 65 years (all p > 0.05) (Table 2). Meanwhile, no correlations of green tea consumption with CSF Aβ42, CSF p-tau, and CSF p-tau/Aβ42 were observed in these two age subgroups (Fig. 2). For subgroup analyses stratified by gender, males with frequent tea consumption had a significantly low level of CSF t-tau/Aβ42 (β= –0.08087, p = 0.049) and a modest trend for decreasing CSF t-tau (β= –0.00520, p = 0.052) (Table 3). No correlations of tea consumption with other CSF biomarkers were observed in two gender subgroups.
Group differences in levels of CSF t-tau and CSF t-tau/Aβ42 across total participants and age subgroups. *represents significant p value obtained from Mann-Whitney U test or t test. CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau.
Group differences in levels of CSF Aβ42, CSF p-tau and CSF p-tau/Aβ42 across total participants and age subgroups. CSF, cerebrospinal fluid; Aβ, amyloid-β; p-tau, phosphorylated tau.
Subgroup analyses of associations between green tea consumption and CSF biomarkers stratified by gender
The β coefficients and p values were calculated using multiple linear regression models after adjustment for age, gender, year of education, APOE ɛ4 status and CM-MMSE. APOE, apolipoprotein E; CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau; p-tau, phosphorylated tau.
Interaction analyses of green tea consumption with APOE ɛ4 status or gender
The two-way ANCOVA results revealed that there were no interaction effects of green tea consumption with APOE ɛ4 status and gender, which thus highlighted the associations of green tea consumption with CSF biomarkers of AD pathology were independent of the effects of APOE ɛ4 status and gender as modifiers (Supplementary Table 1).
DISCUSSION
Accumulating evidence-based research suggests the favorable effects of green tea consumption on cognition and relevant neurodegenerative diseases, while there is still a lack of evidence focused on its correlation with biochemical changes of AD pathology, especially research in humans. In this large-scale study, as expected, we revealed that frequent green tea consumption was associated with low levels of CSF t-tau, independent of the effects of APOE ɛ4 status and gender. Additionally, subgroup analyses presented that green tea consumption negatively associated with CSF t-tau and CSF t-tau/Aβ42 in individuals aged 65 years or younger, as well as associated with CSF t-tau/Aβ42 in males. To the best of our knowledge, this is the first study to assess the association between green tea consumption and CSF biomarkers of AD pathology. The findings are of immense significance in the interventions and therapies targeted AD pathology at early stage using green tea as disease-modified factor.
The CSF Aβ42, CSF t-tau, and CSF p-tau, and their ratios were selected as biomarkers of AD pathology for their best predictive and diagnostic accuracy of cognitive impairment and progression [27–31], as well as the sensitivity to early coincident preclinical changes [32]. Moreover, individuals with rapid change rates of CSF Aβ42 and CSF tau showed faster change rate of Pittsburgh compound B (PiB) mean cortical standardized uptake value ratio and brain atrophy [28, 33]. Longitudinal analyses showed APOE ɛ4 genotype did not affect change rate of CSF Aβ42 over time [34], and no interaction between APOE ɛ4 carrier status and CSF biomarkers were found for change rates of volume loss or cortical thinning [35, 36]. However, some relationships were only revealed in females that baseline low CSF Aβ42 and high CSF t-tau were associated with accelerated rates of hippocampal atrophy and decline in executive function, noteworthily, most associations did not survive Bonferroni correction [37]. Our study thus performed subgroup analyses based on gender, and interestingly, more pronounced associations were observed in males, indicating that potential protective effects may differ in gender. In the present study, there was no interactions for gender or APOE ɛ4 status with green tea consumption. Similar to our findings, previous studies did not find interaction effects of green tea consumption with APOE ɛ4 status and gender on cognitive impairment. A cross-sectional research from Japan reported that the individuals with higher consumption of green tea exhibited lower prevalence of cognitive impairment independent of gender [25]. Consistently, there was no interaction effects of tea intake with gender and APOE ɛ4 on cognitive impairment and cognitive decline [38]. A cohort study comprising 13,645 participants demonstrated that the inverse association of green tea consumption and incident dementia did not modified by the gender [26]. Another longitudinal research found that APOE ɛ4 carriers exhibited accelerated rate of CSF tau accumulation but only in females with abnormal baseline CSF Aβ42 level [39]. This study suggests APOE genotype may produce only subtle effects on downstream pathology, and future studies are needed to replicate due to the trend-level results and the small sample size (27 female APOE ɛ4 carriers and total 239 participants). Moreover, this study applied dichotomous CSF Aβ42 (i.e., abnormal and normal) into the interaction effect analyses and did not consider the interaction of CSF Aβ42 as continuous variable in total participants. These may lead to the differences that our study failed to observe the exacerbated effects of APOE ɛ4 carriers and gender on the relationship between green tea consumption and CSF biomarkers.
Consistent with our study, a research of oligomeric tau model in vitro showed that EGCG, the main polyphenolic constituent of green tea, could inhibit the aggregation of toxic tau oligomers, even at substoichiometric concentrations, thereby alleviating toxicity in neuronal cells [40]. Besides, cadmium-induced neuronal cell deaths in the mouse cortex and hippocampus were apparently inhibited by L-theanine, which were originally isolated from the green tea. Meanwhile, L-theanine could inhibit the activation of glycogen synthase kinase-3β to reduce hyperphosphorylation of tau [41]. Animal experiments revealed that both oral and intraperitoneal EGCG therapies decreased amyloid protein deposition in AD mice, as well as modulated tau profiles thereby improving working memory [42]. However, we failed to identify a correlation of green tea consumption with CSF Aβ42, which might be caused by different disease status, as well as different study subjects. In addition, highlighting the effect of only one constituent of green tea (EGCG) in this animal experiment might contribute to this discrepancy. Recently a review also systematically accumulated similar pieces of evidences about mechanisms that polyphenols, the main constituent of tea, inhibited the self-assembly of tau in vitro. Shred evidence has revealed that tea consumption may decrease the levels of plasma folate [44–47]. But there exist inconsistent conclusions. The small sample sizes (<40) with available plasma folate and vitamin B12 data of this study limited us to assess the association between them and between folate, vitamin B12, and CSF biomarkers. Future research is warranted to clarify whether the tea consumption can modify the AD core protein metabolisms through the folate or vitamin B12. Well-characterized studies have suggested the anti-aging and neuroprotective effects of green tea due to high levels of antioxidants [48, 49]. The increased brain ROS inducing mitochondrial dysfunction and apoptosis causes neurodegenerative diseases, i.e., AD [50, 51]. While tea polyphenols were revealed to scavenge ROS and RNS and induce antioxidant enzymes to bind and chelate excess metals in vitro [52]. Autophagy is an internal process that maintains cellular metabolic homeostasis through the lysosomal degradation and removal of cellular metabolites [48]. The accumulation of amyloid proteins in AD partly attributes to the impaired autophagy [53]. Polyphenols could induce autophagy thereby showing neuroprotective effects [54]. Noteworthily, animal experiments identified that EGCG, the main component of green tea, may be a potential agent against neuroinflammation-associated AD because EGCG could alleviate the neuronal apoptosis as well as the memory impairment induced by lipopolysaccharide [48, 55]. Also, the inhibition effects on activation of astrocytes and pro-inflammatory of microglia, as well as the increased anti-inflammatory expression (such as interleukin [IL]-10) and decrease the pro-inflammatory cytokines (such as IL-1β and tumor necrosis factor [TNF]-α) were observed in the animal models [55–57].
As for biomarkers of neuroinflammation, longitudinal studies showed that CSF IL-10 was associated with rates of longitudinal cognitive decline in MCI, and differed in AD clinical stage [58]. YKL-40, known as chitinase-3-like protein 1 (CHI3L1), is a glycoprotein secreted by astrocytes and infiltrating macrophages. CSF YKL-40 and IL-15 were both increased during the preclinical, prodromal, and dementia stages of AD. High levels of them were associated with increased CSF t-tau, cortical thinning and cognitive deterioration in non-demented individuals [59, 60]. CSF neurofilament light (NFL), as a neuronal cytoskeletal protein associated with axonal injury, was associated with the dementia stage of AD and CSF tau biomarkers (t-tau and p-tau) [58]. Plasma NFL in association with AD diagnosis, cognition, AD-related atrophy, and hypometabolism implies its potential usefulness in clinical trials as a noninvasive biomarker in AD [61, 62]. Therefore, future research can further assess the associations of green tea consumption with these CSF and plasma biomarkers of specific molecular mechanisms in AD developments, thereby better complementing the effects of green tea in neurodegeneration.
Several limitations should be considered in the interpretation of main findings. First, conclusions deriving from observational studies could not clarify a necessary causality because of lack of a temporal relation between tea consumption and CSF biomarkers. Future research studies are warranted to track the trajectories of biochemical changes and test the intervention effects of green tea consumption on clinical outcomes. Second, individual frequency of green tea consumption based on a self-reported questionnaire rather a regular record about daily intake of tea might be subjective and rough. The records of duration time and daily amount of tea consumption would facilitate quantitative analyses of future cohort studies. Of course, because of frequent consumption of green tea in Chinese social activities and more favorable effects of green tea on cognition, our study did not consider correlations of other types of tea (such as black and oolong tea) with CSF biomarkers. Maybe future prospective studies that distinguish the effects of tea types and the diverse constituent of green tea on AD biological changes would complement existing understandings about underlying protective mechanisms.
Notwithstanding its limitation, this study does uncover several novel and significant findings. Here, we used a well-established study of cognitively intact individuals and performed a careful and systematic analysis. As such, we avoided reaching some conclusions driven by confounding effects of complex AD status and poor individual’s daily living ability. In addition, all analyses were performed on participants from an ongoing epidemiological CABLE cohort of the Chinese Han population. The findings reflected the features of green tea consumption in the background of specific demographic factors, and homogeneous data provided more credible conclusions. Of course, the results of subgroup analyses depending on demographic factors need further large-scale prospective studies to validate. In brief, by teasing out green tea consumption associated early changes of AD pathological biomarkers, our study provided novel clues in need for longitudinal research and clinical trials about prevention and drug development thereby slowing or preventing disease process.
In conclusion, our study suggests that green tea consumption may mitigate the detrimental effects of CSF biomarkers of tau pathology in cognitively intact individuals. Individuals aged 65 years or younger and males probably benefit more from such modifiable lifestyle factors. The constituents of green tea may improve abnormal tau metabolism and are promising targets in interventions and drug-therapies. Future prospective studies that expand the sample size and distinguish diverse constituents of green tea on AD biological changes would complement existing understandings about underlying mechanisms.
Footnotes
ACKNOWLEDGMENTS
The authors thank all participants of the present study as well as all members of staff of the CABLE study for their role in data collection.
This study was supported by grants from the National Natural Science Foundation of China (91849126, 81571245, and 81771148), the National Key R&D Program of China (2018YFC1314700), Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01), and ZHANGJIANG LAB, Tianqiao and Chrissy Chen Institute, and the State Key Laboratory of Neurobiology and Frontiers Center for Brain Science of Ministry of Education, Fudan University.
