Study of telomere length and different markers of oxidative stress in patients with Parkinson's disease

Abstract

Background

Many studies have shown that short telomere length (TL) is associated with high oxidative stress and various age-related diseases. Parkinson's disease (PD) is an age-related disease, and although its pathogenic mechanism is uncertain, oxidative stress is believed to be implicated in this pathology. The aim of this case-control study was to assess both TL and the different markers of oxidative stress in elderly patients with PD compared to age control subjects.

Methods

20 PD patients and 15 age-matched controls, >65 years were studied. TL was measured by Southern blotting from DNA samples extracted from white blood cells. Superoxide dismutase (SOD) activity and plasma levels of total glutathione and protein carbonyls were determined.

Results

There was a trend for lower TL in PD patients: 6.06 ± 0.81 kb in PD versus 6.45± 0.73 kb in controls (p = 0.08). No significant difference was found between the two groups in terms of oxidative stress markers. In controls, age was the main determinant of telomere shortening (r = −0.547; p = 0.03) whereas, in PD patients, telomere shortening was mainly dependent on plasmatic concentrations of carbonyl proteins (r= −0.544; p=0.044). In PD patients, a negative association was observed between plasma carbonyl protein levels and SOD activity (r= − 0.622, p=0.004).

Conclusions

In PD, TL is shorter in presence of high oxidative stress as measured by carbonyl protein levels. The absence of telomere attrition with age among patients with PD could reflect a telomere regulation by mechanisms other than age.

Introduction

Telomeres are tandem repeats of TTAGGG of the DNA sequence at the ends of eukaryotic chromosomes. With each cell replication, telomeres shorten (attrition) until reaching a critical size where all cell replication becomes impossible, hence inducing cellular senescence (1, 2). At a given age, telomere size depends primarily on their size at birth and on the chronic effects of oxidative stress, the latter currently considered as the most powerful factor in the attrition of telomere size. Experimental evidence, obtained from various cell types (3, 4, 5), indicate that oxidative stress accelerates the attrition of telomeres. Thus, telomere length is both an indicator of the “life history” of the cell and of its fate (6). In humans, many studies have shown that relative telomere attrition is associated with various age-related diseases including hypertension, arterial stiffening, atherosclerosis and Alzheimer's disease (7, 8, 9, 10, 11). Parkinson's disease (PD) is an age-related neurodegenerative disease, and although its pathogenic mechanism is uncertain, oxidative stress is believed to be implicated in this pathology (12, 13). Therefore, one could expect that PD patients would show increased markers of oxidative stress and shorter telomeres. However, few data exist regarding telomere length and PD. In a case-control study (96 cases and 172 age-matched controls), telomeres were not associated with the risk of PD (14). In a recent study by Guan et al. (15), although the authors did not observe a statistical difference in mean telomere length of peripheral leukocytes between PD patients and control participants, a percentage analysis of telomere length suggested that telomere shortening was accelerated in PD patients in comparison to controls (15).

To our knowledge, there has yet to be a study investigating the relationship between telomere attrition and oxidative stress in PD. The aim of the present case-control study was to assess both telomere length and various markers of oxidative stress in elderly patients with PD compared to age control subjects.

Methods

Population

This study is a sub-study of the project described previously (16). The present study included 20 patients with PD diagnosed according to the UK PD Society Brain Bank criteria, and 15 age-matched healthy controls, aged 65 years and older. All study participants were consecutively admitted to the geriatrics day hospital of Nuremberg and fulfilled the criteria of the SENIEUR protocol (17). This protocol defines healthy aged individuals for immunogerontological studies by setting clear health guidelines based on clinical and laboratory data. Briefly, the following exclusion criteria were applied to all participants: abdominal hematological and hepatic values, current myocardial infarction and stroke, cardiovascular disease, hypertension, diabetes mellitus, hyperuricemia, current infections and inflammation (defined as a serum C-reactive protein (CRP) value > 10 mg/L), malignancy, malnutrition and alcoholism. Also excluded were subjects with other neurodegenerative diseases, those on special diets, or taking antioxidant supplements or drugs with known influence on the immune system. Participants with prescribed antiparkinsonian drugs, acetylsalicylic acid (100 mg) or statins were included. All PD patients were treated with antiparkinsonian drugs, including levodopa, direct dopaminergics, amantadine, catechol-O-methyl transferase inhibitors, and monoamine-oxidase—B-inhibitors. Reasons for admission to the geriatric day hospital were adjustment of antiparkinsonian medication and physiotherapy in patients with PD, or treatment of depression or chronic pain syndrome in controls. All subjects gave written informed consent before entering the study. The study protocol was approved by the Ethics Committee of the University of Erlangen-Nuremberg, Germany.

Analytical methods

Blood was collected between 9.00 am and 10.00 am after an overnight fast of at least 12 h. Serum and EDTA samples were obtained following centrifugation at 1800 x g for 15 min, and stored at -28 C until analysis. Serum albumin was measured nephelometrically using an Immage Nephelometer (Beckman-Coulter Ltd., Krefled, Germany). The Olympus AU2700 Clinical Chemistry System and manufacturer's reagents (Olympus, Hamburg, Germany) were used to measure lipids (total cholesterol, HDL cholesterol and triglycerides). LDL cholesterol was calculated using the Friedwald formula. CRP concentrations were determined with the Dynamik CRP AU kit (Biomed, Munich, Germany).

Measurements of the Terminal Restriction Fragments (TRF) Length

DNA samples were extracted from white blood cells (WBCs) as previously described (18) and verified for integrity on 0.8% agarose gel. TRF length was measured as previously described (7, 8, 19). Briefly, DNA samples were digested overnight with restriction enzymes HinfI and RsaI (40 units) and resolved on a 0.8% agarose gel (15 cm x 25 cm) at 40 V (PowerPac Basic, Bio-Rad). After 20 h, the DNA was depurinated, denatured and neutralized and then transferred for 1.5 h onto a positively charged nylon membrane (Amersham). The membranes were hybridized with the telomeric probe [digoxigenin 3'-end labeled 5'-(CCTAAA) 3] overnight and washed in a sodium chloride and sodium citrate buffer. The digoxigenin-labeled probe was detected by the digoxigenin luminescent detection procedure (Roche) and exposed on X-ray film. Each DNA sample was measured in duplicate.

Oxidative stress determination

All oxidative stress markers were determined using the Cayman assay kits according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI).

Briefly, carbonyl groups react with 2, 4-dinitrophenylhydrazine (DNPH) to generate chromophoric dinitrophenylhydrazones. Proteins were precipitated with trichloracetic acid (20%) and washed with ethanol/ethyl acetate mixture. The resulting protein precipitates were dissolved in guanidine - HCl solution and the absorbances measured at 370nm, using a molar extinction coefficient of DNPH (e = 2.2 104L.mol-1cm-1). Protein contents were determined simultaneously in the HCl blank pellets and protein carbonyl content was expressed in nanomoles per milliliter (nmol/ml).

For the determination of plasma total glutathione (GSH) content, plasma samples were deproteinized with metaphosphoric acid (50 mg/mL) and concentrated 3-fold. The sulfhydril group of GSH reacts with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) via GSH reductase and produces a yellow-colored reaction product TNB (5-thio-2-nitrobenzoic acid) with Ellman's reagent. The mixed disulfide GSTNB, which is concomitantly produced, is reduced by GSH reductase to recycle GSH and produce more TNB. Absorbance was measured at 405 nm using a microplate reader.

Finally, assay temperature for Superoxide Dismutase (SOD) enzymatic activity was 25°C. Using a 96 well plate, radical detector (Tetrazolium) was added to sample and SOD standard wells. The reactions were initiated by adding Xanthine Oxidase to all wells. After incubation at room temperature, the absorbance was measured at 450 nm using a microplate reader. This kit utilizes a Tetrazolium salt for detection of superoxide radicals generated by Xanthine oxidase. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.

Statistical analysis

All statistical analyses were carried out with the NCSS (version 2000) software. All variables were tested for normal distribution of the data. Differences between the groups of patients with and without PD were examined using the Student's t test or for continuous variables, while Chi-Square test was used for categorial data. Bivariate correlations were performed using the Pearson correlation coefficient. A P value = 0.05 was considered as significant.

Results

Main demographic, clinical and laboratory parameters for patients with PD and for age-matched healthy controls are summarized in Table 1. There was a trend for lower TRF in PD patients: 6.06 ± 0.81 kb in PD versus 6.45± 0.73 kb in controls (p = 0.08 after adjustment for age and gender). No significant difference was found between the two groups with regard to plasmatic SOD activity, plasmatic concentrations of carbonyl proteins or total GSH (p= 0.38; p= 0.35; p= 0.84; respectively).

Table 1.

Demographic, clinical and laboratory data of the study population

	PD Patients n=20	Controls n=15	P-Value
Demographic, clinical data Age, years	77.5 ± 6.0	78.7 ± 8.6	0.62
Gender, n, male/female	11/9	4/11	0.09
Weight, kg	76 ± 17.5	67.5 ± 11.7	0.10
Height, cm	165.2 ± 12.5	160.5 ± 9.1	0.22
BMI, kg/m²	27.6 ± 4.2	26.4 ± 5.2	0.48
CV history, n, %	3, 15%	0, 0%	0.12
Smoking, n, %	0, 0%	2, 13%	0.09
Statins, n, %	4, 20%	4, 27%	0.56
Acetylsalicylic acid, n, %	10, 50%	6, 40%	0.68
Stage of PD	3.6 ± 0.7	n.a
Duration of PD, year Laboratory data	4.4 ± 2.8	n.a
Mean TRF, kb	6.03 ± 0.81	6.45 ± 0.73	0.11
SOD, U/ml	0.248 ± 0.04	0,261 ± 0,05	0,38
Total GSH, μM	1.85 ± 0.77	1.80 ± 0.46	0.84
Carbonyl Proteins, nmol/ml	94.1 ± 29.9	102.6 ± 23.1	0.35
TG, mmol/L	1.52 ± 0.50	1.50 ± 0.72	0.92
Cholesterol, mmol/L	5.17 ± 1.07	5.30 ± 0.89	0.70
HDL-C, mmol/L	1.46 ± 0.44	1.60 ± 0.32	0.30
LDL-C, mmol/L	3.01 ± 0.86	3.02 ± 0.75	0.99
Protein, g/L	70.9 ± 3.9	71.3 ± 4.5	0.80
Albumin, g/L	40 ± 3.3	40.5 ± 3.3	0,67

Data are expressed as: Mean±SD and number (n) /Percentage (%). Student T-test (continuous variables) or Chi-Square tests (categorical variables) were used to verify the probability level between the two groups. PD: Parkinson's Disease; BMI: Body Mass Index; CV: CardioVascular, TRF: Terminal Restriction Fragment; SOD: Superoxide dismutase; Total GSH: Total Glutathione; TG: Triglycerides; HDL-C: High-Density Lipoprotein Cholesterol; LDL-C: Low-Density Lipoprotein Cholesterol; n.a: not applicable.

Effect of age on TRF length and oxidative stress markers in both groups

The relationship between TRF length and age was studied in both PD patients and age-matched controls. Telomere shortening with aging was observed only in controls (r = -0.547; p = 0.03), whereas in PD patients, no significant correlation was detected with age (r = 0.073, p = 0.76) (fig. 1).

Figure 1.

Association between TRF length and age in PD patients and healthy controls kb: kilobase; PD: Parkinson's Disease; TRF: Terminal Restriction Fragment

In PD patients but not in controls, a negative relationship was found between SOD activity and age (r = -0.522; p=0.02), whereas no other significant association was found between other oxidative stress markers and age in PD patients. In contrast, there was no correlation between age and any of the oxidative stress markers in controls.

Analysis of the relationship between oxidative stress markers and TRF length

In PD patients, a significant negative association was observed between the plasmatic concentrations of carbonyl proteins and TRF length (r= -0.544; p=0.044) (fig.2). This association remained unchanged after adjustment for age. In contrast, no other significant association was found between other oxidative stress markers and TRF length in PD patients, nor between any of the oxidative stress markers and TRF length in healthy controls (data not showed). Finally, plasmatic SOD activity was negatively correlated with plasmatic concentrations of carbonyl proteins (r= -0.622, p=0.004) (fig.3).

Figure 2.

Association between Carbonyl Proteins and TRF in PD patients and healthy controls SOD: Superoxide dismutase; PD: Parkinson's Disease

Figure 3.

Association between Carbonyl Proteins and SOD activity in PD patients and healthy controls SOD: Superoxide dismutase; PD: Parkinson's Disease

Discussion and conclusion

To our knowledge, this is the first study concomitantly measuring telomere length and peripheral markers of oxidative stress in PD patients. The present case-control study conducted in a limited number of individuals provides some first elements of an implication of oxidative stress in the attrition of telomere length in PD patients. Notably, two results point in this direction: a trend toward lower TRF length (p=0.08) and a significant inverse relationship between plasmatic concentrations of carbonyl proteins and TRF length.

The difference in telomere length of 0.4 kb observed between the two groups is comparable to differences reported in previous studies between controls and patients with different age-related alterations and diseases such as aortic stiffness (8, 9) carotid atherosclerosis (7) and dementia (10, 20). However, the association between PD and telomeres had never been established. For instance, Wang et al. (14) found no association between shorter telomeres with an increased risk of PD. In another study addressing the issue of telomere attrition in PD patients (15), Guan et al. did not observe a statistical difference in mean telomere length of peripheral leukocytes between PD patients and control participants. They did however observe mean telomere lengths shorter than 5 kb in only the PD patients and a significant PD-associated decrease in telomeres with a length ranging from 23.1 to 9.4 kb in patients in their 50s and 60s suggesting that telomere shortening is accelerated in PD patients in comparison to the normal population. Nevertheless, due to the small size of their population (28 Japanese male PD patients), we believe that this percentage analysis of telomere length would necessitate confirmation through large-scale studies.

Indeed, results herein could be influenced by intrinsic factors such as apoptosis which, according to Lev et al (21), play a role in the pathology of PD. Since apoptosis is involved in the removal of senescent cells and these cells have short telomeres, it is likely that apoptosis interferes with the overall average length of telomeres.

The presence of a correlation between TRF and age observed only in controls but not in PD patients is another interesting result of the present study. The slope of the relationship in the control group indicates an annual attrition of 47 bases per year which is comparable to that reported in other studies (7, 22). Conversely, the absence of telomere attrition with age among patients with PD could reflect telomere regulation by mechanisms other than age; i.e. the effects of PD outweigh the influence of age. However, given the small size of the population of the present study, these results should be interpreted with due caution.

Despite the fact that oxidative stress has been proposed to play an important role in the pathogenesis of PD (12, 13, 23), no significant differences in markers of oxidative stress, namely plasma total GSH, protein carbonyls and SOD activity, were observed in the present study between PD patients and healthy controls. It is possible that the lack of such difference may be at least partially related to the fact that all patients were treated with l-dopa which may have anti-oxidant effects as previously proposed (24, 25).

In agreement with Serra et al. (26) and Younes-Mhenni et al. (27), we noted no significant changes in total glutathione levels in PD patients compared to controls. However, it should be kept in mind that one of the earliest biochemical changes seen in PD is a reduction in the levels of total glutathione, a key cellular antioxidant (28).

Alam et al. (29) suggested that oxidative protein damage, as measured by carbonyl assay in postmortem brain tissue from patients with PD and age-matched controls, is not a major contributor to the early stages of PD. Sudha et al. (30) also indicated no significant change in erythrocyte SOD activity of PD patients, although previous peripheral studies of SOD activity have been discordant. Indeed, an increase in peripheral blood SOD activity has been shown by some authors (27, 31), whereas others reported a significant decrease in SOD activity in the blood of PD patients compared to controls (32, 33). Moreover, in agreement with Ihara et al. (34), our analysis showed a negative correlation between SOD activity and age in PD patients, suggesting a decrease in anti-oxidant capacities with aging.

Thus, the present results indicate the presence of an imbalance between antioxidant defense (Plasma SOD activity) and production of oxidation (protein carbonyl levels) only in PD patients. The negative relationship between TRF length and protein carbonyls strongly suggests that this “imbalance” may play an important role in telomere attrition. This “clinical” result corroborates previous reported data from in vitro (35) and in vivo (3) studies indicating that oxidative stress is responsible for accelerated telomere attrition. This negative association between oxidative stress and TRF length was described in other chronic age-related diseases. Matthews and colleagues suggested that human atherosclerosis is characterized by senescence of vascular smooth muscle cells, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening (4). Moreover, we have recently shown that TFR is associated with oxidative stress as measured by plasma Nitrotyrosine levels in male with type 2 diabetes (19).

This study is not without limitations. Firstly, the present study population is relatively small. Secondly, although it has been suggested that TRF measurement in easily accessible tissues such as peripheral blood leukocytes could serve as a surrogate parameter for relative telomere length in other tissues (36), oxidative stress markers in plasma may not reflect accumulated oxidative stress involved in the pathology of PD. Thirdly, large-scale, longitudinal cohorts are necessary to elucidate how telomere shortening is accelerated in an oxidative stress environment.

In conclusion, the present case-control study indicates that in Parkinson's disease, TRF length is shorter in presence of high oxidative stress as measured by carbonyl protein levels. The absence of telomere attrition with age among patients with PD could be explained by the fact that the effects of oxidative stress overwhelm the influence of age.

Acknowledgments:

We thank Mr Pierre Pothier for his very pertinent comment on this paper and for linguistic corrections.

Financial disclosure: None of the authors had any financial interest or support for this paper.

Abbreviations

BMI

Body Mass Index

CRP

C-Reactive Protein

CV

CardioVascular

DNPH

2, 4-dinitrophenylhydrazine

DTNB

5,5′-dithiobis-2-nitrobenzoic

GSH

Glutathione

HDL-C

High-Density Lipoprotein Cholesterol

kb

kilobase

LDL-C

Low-Density Lipoprotein Cholesterol

n.a

not applicable

PD

Parkinson's Disease

SOD

Superoxide dismutase

TG

TriGlycerides

TL

Telomere Length

TNB

5-thio-2-nitrobenzoic

TRF

Terminal Restriction Fragment

WBCs

White Blood Cells