- © 2016 by the Association of Clinical Scientists, Inc.
Telomere Shortening in Alzheimer’s Disease Patients
- Mengyuan Liu1,2,4,
- Ya Ruth Huo3,
- Junwei Wang1,
- Change Wang1,
- Shuling Liu1,
- Shuai Liu1,
- Jinhuan Wang1,2 and
- Yong Ji1,4⇑
- 1Department of Neurology, Tianjin HuanHu Hospital, Tianjin, China
- 2Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Tianjin HuanHu Hospital, Tianjin, China
- 3School of Medicine, University of New South Wales, Kensington, NSW, Australia
- 4Tianjin Dementia Institute, Tianjin Huanhu Hospital, Tianjin, China
- Address correspondence to Yong Ji, MD., PhD., Tianjin Huanhu Hospital, Qixiangtai Road 122, Hexi, Tianjin 300060, China; phone: +86 022 60367885; e mail: jiyongusa{at}126.com
Abstract
Background and Aims Sporadic Alzheimer’s Disease (AD) is considered an age-related disease, and telomere length has been shown to decrease with age in animal and human studies. Shortening of telomere length has also been reported to beis associated with numerous neurodegenerative diseases, including AD. The aim of this study is to confirm and further elucidate the relationship between peripheral telomere length and Alzheimer’s disease, cognitive function, and duration of Alzheimer’s disease.
Methods In this study, we recruited 165 Han Chinese subjects, consisting of 79 normal controls and 86 patients with AD without family history of AD or vascular dementia cases. We measured telomere length (T/S ratio) at baseline using quantitative PCR.
Results Our data showed that the negative correlation between telomere length and age become much stronger in AD patients (n=86, r=−0.382, p<0.001) compared to the control group (n=79, r=−0.024, p=0.833), presenting an estimated telomere loss rate of 0.003 T/S ratio/year (p<0.001). Moreover, we observed that the duration of AD is significantly negatively correlated with telomere length (n=86, r=−0.224, p=0.039), especially in female patients (n=43, r=−0.375, p=0.013) and ApoE4-noncarrier patients (n=52, r=−0.368, p=0.007). We also had some unexpected results, namely that in AD patients, higher Minimum Mental State Examination (MMSE) score were associated with shorter telomere length (n=76, r=−0.281, p=0.014).
Conclusions This study revealed an accelerated rate of telomere shortening in AD, as well as strong influence of gender and ApoE status on telomere length in the context of this disease.
Introduction
Alzheimer’s disease (AD) is the most common form of dementia in elderly populations worldwide, characterized by a progressive loss of executive function, visuospatial cognition, and memory. These changes are most likely a result of the degeneration of cholinergic neurons in the ascending cholinergic pathways of the basal forebrain [1], cells which are crucial to memory and cognition [2].
Telomeres are special chromatin structures located at the ends of chromosomes. They comprise tandem DNA sequence repeats and specific bounding proteins. Telomeres play key roles in protecting chromosomes from degradation and fusions. Telomeres shorten as cells divide and have been reported to decrease in length with age in animal and human studies [3]. Reduced telomere length has been documented to be associated with Alzheimer’s disease. A multiethnic community-based study revealed that shortened leukocyte TL is associated with risks for dementia and mortality, and shorter TL was a risk for earlier onset of dementia in females [4]. Results from a recent Mendelian Randomization (MR) Study demonstrated that shorter TL was causally associated with a higher risk for AD [5]. This is somewhat unsurprising considering that sporadic AD is an age-related disease, and many age-related diseases have been found to be strongly associated with reduced telomere length. Furthermore, telomeres are especially sensitive to radical oxygen species (ROS). Even in non-dividing cells that have accumulated ROS, the process of telomere shortening is accelerated [6]. Numerous studies have also reported factors that relate to telomere length, such as physical activity [7], tobacco smoking [8], alcohol consumption [9], and even paternal age [10]. These factors have also been found to be associated with Alzheimer’s disease; change in telomere length could be the missing link.
The aim of this study is to confirm and further elucidate the relationship between peripheral telomere length and Alzheimer’s disease, cognitive function, and duration of Alzheimer’s disease. Stratification for apolipoprotein E (ApoE) status and gender will be undertaken.
Materials and Methods
Subjects
We recruited 165 Han Chinese subjects consisting of 86 patients with AD and 79 normal controls (NCs). All patients with AD were diagnosed with probable AD based on the National Institute of Neurological and Communicative Disorders and Stroke – Alzheimer’s Disease and Related Disorders Associations (NINCDS-ADRDA) at the Dementia Center at Tianjin Huanhu Hospital in Tianjin City, China [11]. Exclusion criteria for members of the control group included (i) a family history of AD and (ii) vascular dementia cases (a score over 2 points on the Hachinski ischemic scale) [12]. Age-matched control subjects exhibited no symptoms of dementia and had an MMSE score higher than 23. Blood samples were collected from all participants. This study was approved by the Ethics Committee of the Huanhu Hospital. Written informed consent was obtained from both the patients and their caregivers.
Telomere measurement by quantitative real-time PCR
The genomic DNA of every subject was isolated from peripheral nuclear blood cells using the Omega Blood DNA Kit (Omega Biotek, Inc.) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was done on the Roche Lightcycler II using telomeric primers(Tel), and primers for a reference control gene (the human 36B4 single-copy gene). For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA from the same tissues. The telomere signal was normalized to the signal from the single-copy gene to generate a T/S ratio indicative of relative telomere length [13].
The primer oligonucleotides used to amplify the telomere sequence (Tel) and the house keeping gene (36B4) are presented in Table 1. The qPCR was performed in a total volume of 20μL, containing 5μL DNA template (4ng/μL), 12.5μL Syber Green Mix (TIANGEN BIOTECH CO. LTD.), 2μL/2.6μL of each primer (Tel/36B4, 10μM) and deionized distilled water (ddH2O). Thermal cycling conditions were set at 94°C for 10 min followed by 35 cycles of 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, with final extension for 5 min at 72°C [14]. APOE was genotyped by the RFLP method previously described in a published paper [15].
Forward and reverse primer oligonucleotides used to amplify the telomere sequence (Tel) and the housekeeping gene (36B4)
Statistical analysis
We calculated the length of disease period in AD patients by using the current age minus the onset age of AD. Then, the Spearman correlation coefficient was analyzed to determine the association between telomere length and length of disease period in AD patients.
We surveyed the MMSE score of each subject and then performed the Pearson correlation analysis. The group of normal controls was selected according to their ages and MMSE scores, which were supposed to be above 23.
Results
The baseline characteristics of NCs and AD groups are illustrated in Table 2. The two groups show similar telomere length and gender distribution. Also, there is no statistically significant difference in telomere length between males and females, or ApoE4 carriers and ApoE4 non-carriers, in either group.
Baseline characteristics of controls and those with Alzheimer’s disease.
In the total group, telomeres shortened with increasing age (n=165, r=−0.231, p=0.003). The association between telomere length and age became more significant in the AD group (n=86, r=−0.382, p<0.001), where linear regression analysis showed a yearly telomere loss of 0.003 (T/S ratio) (p<0.001). In the NCs, there was no significant association between telomere length and increasing age (n=79, r=−0.024, p=0.833) (Figure 1).
T/S ratio value of telomere length and age of normal controls and AD patients.
Telomere length was negatively correlated with duration (years) of having AD (n=86, r=−0.224, p=0.039). When stratified for sex, the association between telomere length and length of disease period was greater and remained significant in females (n=43, r=−0.375, p=0.013) (Figure 2B), but became non-significant in males (n=43, r=−0.137, p=0.382) (Figures 2A & 4A). When stratified for ApoE ε4 status, the association between telomere length and length of disease period was stronger and remained significant in ApoE ε4 non-carriers (n=52, r=−0.368, p=0.007) (Figure 2C), but non-significant in ApoE ε4 carriers (n=34, r=−0.038, p=0.833) (Figures 2D & 4B).
Distribution of relative telomere length (T/S ratio) corresponding to disease duration time (years) of AD patients in comparison of males (A) and females (B), and in comparison of ApoE4 noncarriers (C) and ApoE4 carriers (D).
Among AD patients, there was a negative association between telomere lengths and MMSE scores (n=76, r=−0.281, p=0.014). When further stratified for sex, the negative correlation became stronger and remained significant in males (n=38, r=−0.424, p=0.008) (Figure 3A) but non-significant in females (n=38, r=−0.116, p=0.488) (Figures 3B & 4C). When stratified for ApoE ε4 status, the negative correlation became stronger and remained significant for ApoE ε4 carriers (n=33, r=−0.382, p=0.028) (Figure 3D), while it was non-significant in ApoE ε4 non-carriers (n=43, r=−0.202, p=0.194) (Figures 3C & 4D). No correlation was found between MMSE score and telomere length in NCs overall or when stratified for gender or ApoE ε4 status (n=79, r=0.015, p=0.894). No association was found between MMSE score and length of disease period overall and when stratified by gender and ApoE ε4 status.
Distribution of relative telomere length (T/S ratio) corresponding to MMSE score of AD patients in comparison of males (A) and females (B), and in comparison of ApoE4 noncarriers (C) and ApoE4 carriers (D).
Distribution of telomere length corresponding to MMSE score or length of disease period, illustrated in scattered dots. Distribution of relative telomere length (T/S ratio) corresponding to disease duration time (years) of AD patients in comparison of males and females (A), and in comparison of ApoE4 noncarriers and ApoE4 carriers (B). Distribution of relative telomere length (T/S ratio) corresponding to MMSE score of AD patients in comparison of males and females (C), and in comparison of ApoE4 noncarriers and ApoE4 carriers (D).
Discussion
It is widely acknowledged that accelerated telomere shortening has been observed in numerous neurodegenerative diseases, such as Down syndrome [16], dementia with Lewy bodies [17], and Alzheimer’s disease [18–20]. A recent study showed that in elderly people, shorter telomere length indicate increased risk of amnestic mild cognitive impairment (aMCI) [21]. The inverse correlation between telomere length and age has been verified in our AD participants. Linear regression yielded an estimated telomere loss rate of 0.003 T/S ratio/year (p<0.001), and the Pearson correlation coefficient between telomere length and age is −0.382 (p<0.001). However, the rate of telomere length loss in our study was slower than the average loss rate (0.010 T/S ratio/year) seen in previous studies [22]. Unlike those with AD, those who were cognitively normal showed no significant decrease of telomeres over 60 to 70 years. The slower rate of telomere loss presented in this study may due to the specific and relative narrow age range of the subjects.
This study found that increases in the years of being affected by AD is significantly correlated with decreases in telomere length in all AD patients. However, further stratification found it to only remain significant in females and ApoE ε4 non-carriers. This suggests that the ApoE ε4 carrier status or the male gender is a protective factor against telomere shortening in AD. Thus, we supposed that the disease condition may provide a detrimental context for telomere length maintaining. However, as we were not able to measure the patients’ telomere length when they were just affected with AD, further longitudinal studies are necessary to elucidate this association more explicitly.
Our findings also show a negative association between telomere length and MMSE score in AD patients, i.e. as telomere length decreased, MMSE score increased. Further analysis revealed that this remained significant in males and ApoE ε4 carriers, but was not significant in females and ApoE ε4 non-carriers. These findings highlight the importance of controlling for gender and ApoE status. A previous study in an AD mouse model found that telomere shortening improved the spatial learning ability and reduced amyloid plaque pathology [11]. In contrast, numerous studies in human populations have reported that telomere length decreases with cognitive decline in both elderly individuals [23] and those with AD [6,20,24]. Studies on the association of telomere lengths and cognitive function in have revealed contrary results. Further studies are warranted to examine the association between telomere length and MMSE scores, and should be controlled for gender and ApoE4 status [25].
The limitations of our study must be taken into account when assessing our findings. In this study, we analyzed the peripheral white blood cells of each subject due to easy availability. Changes of blood cell telomere length might not reflect changes of brain cell telomere length, and thus the correlation between telomere length and neuron function might be indirect. It has been reported that patients with AD exhibit an accelerated telomere loss in peripheral blood cells [18,26,27], whereas longer telomere lengths were observed from hippocampus cells compared to control samples [27]. Secondly, the sample size was comparatively small and requires further validation with larger participant groups. However, as we could detect the significant association for males and ApoE ε4 carriers for MMSE with telomere length, as well as females and ApoE ε4 noncarriers for disease duration with telomere length, our sample was large enough for the purpose of this study.
In conclusion, this study highlights the accelerated rates of telomere shortening in AD patients. However, the lack of consistency in literature regarding telomere length and AD suggests that it is not yet ready to serve as a general biomarker to identify those at risk of AD. If peripheral white cells’ telomere lengths are to be used to diagnose or assess Alzheimer’s disease, long-term prospective studies are warranted. This study also identifies the strong influence of gender and ApoE status on telomere length. These findings make it necessary for future telomere studies to control for these two variables.
