J Neurol (2010) 257:540–545 DOI 10.1007/s00415-009-5357-2
ORIGINAL COMMUNICATION
Serum levels of brain-derived neurotrophic factor correlate with motor impairment in Parkinson’s disease Paula Scalzo • Arthur Ku¨mmer • Thales Lage Bretas Francisco Cardoso • Antoˆnio Lu´cio Teixeira
•
Received: 27 March 2009 / Revised: 1 October 2009 / Accepted: 8 October 2009 / Published online: 22 October 2009 Ó Springer-Verlag 2009
Abstract The brain-derived neurotrophic factor (BDNF) is a potent inhibitor of apoptosis-mediated cell death and neurotoxin-induced degeneration of dopaminergic neurons. There is a growing body of evidence implicating BDNF in the pathogenesis of Parkinson’s disease (PD), suggesting it may eventually be used in the development of neuroprotective therapies for PD. The serum BDNF of 47 PD patients and of 23 control subjects was assessed, and serum BNDF levels were significantly decreased in PD patients when compared with controls (p = 0.046). Interestingly enough, BDNF correlated positively with a longer time span of the disease, as well as with the severity of the PD symptoms and with more advanced stages of the disease. Additionally, higher BDNF levels also correlated with poor balance as assessed by the Berg Balance Scale, more time spent at the Timed Up & Go Test, reduced speed of gait and shorter distance walked during the Six-Minute Walk Test. Our results corroborate the literature regarding the P. Scalzo A. Ku¨mmer T. L. Bretas A. L. Teixeira Laboratory of Immunopharmacology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil A. Ku¨mmer T. L. Bretas A. L. Teixeira Neuropsychiatric Branch, Department of Internal Medicine, School of Medicine, UFMG, Belo Horizonte, Brazil F. Cardoso Movement Disorders Clinic, General Hospital, UFMG, Belo Horizonte, Brazil A. L. Teixeira (&) Laborato´rio de Imunofarmacologia, Departamento de Bioquı´mica e Imunologia (ICB), Universidade Federal de Minas Gerais, Av. Antoˆnio Carlos, 6627, Bloco O4, Sala 202, Pampulha, 31270-901 Belo Horizonte, Brazil e-mail:
[email protected]
123
involvement of BDNF in PD. We hypothesize that lower BDNF levels in early stages of the disease may be associated with pathogenic mechanisms of PD. The increase of BDNF levels with the progression of the disease may be a compensatory mechanism in more advanced stages of PD. Keywords Parkinson’s disease Brain-derived neurotrophic factor Neurotrophins Neurodegeneration Depression Motor impairment
Introduction Parkinson’s disease (PD) is a chronic neurodegenerative disease characterized by a progressive and irreversible loss of the dopaminergic neurons of the substantia nigra pars compacta [1]. The etiology of the degeneration of SNpc cells is unknown. It has been proposed that the neurodegenerative process may result from an insufficient supply of neurotrophic factors. The neurotrophin family is a group of small, basic, secreted proteins that aid in the differentiation, survival and maintenance of specific neuronal populations. This family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophins (NT) 3, 4/5 and 6. BDNF supports the survival and the differentiation of dopaminergic neurons. Also, BDNF is a potent inhibitor of apoptosis-mediated cell death and neurotoxin-induced degeneration of dopaminergic neurons, which suggests that it is likely to be used in the development of neuroprotective therapies for PD [2]. Many studies have documented some evidence of a decreased expression of BDNF in different neurodegenerative diseases [3, 4]. The decreased BDNF protein has also been detected in the substantia nigra in PD, especially in
J Neurol (2010) 257:540–545
the ventro-lateral tier [5, 6]. Analyses of brain tissue from PD patients showed significantly lower BDNF concentrations in nigrostriatal dopamine regions than in the brain tissue of control subjects [7]. On the other hand, glial cells of the substantia nigra of PD patients expressed increased levels of BDNF, possibly in response to signals released from the failing neurons [8]. Several genetic studies have demonstrated a possible correlation between BDNF gene polymorphisms and PD [9]. In 6-OH-dopamine (6-OHDA) rat model, intrastriatal grafts of fibroblasts genetically engineered to produce BDNF partially prevented the loss of nerve terminals and completely prevented the loss of cell bodies of the nigrostriatal dopaminergic pathway [10]. Neurotoxins did not decrease the levels of BDNF in the substantia nigra, suggesting that an up-regulation of BDNF synthesis by pharmacological means may be a viable therapy to slow down the progress of PD and other neurodegenerative diseases [11]. Experimental evidence from animal models suggests that circulating BDNF levels may reflect BDNF levels in the brain [12]. However, BDNF levels in the serum of PD patients have not been assessed up to now. This technique raises the possibility of correlating BDNF levels with different clinical aspects of PD in an easier manner than postmortem studies. The objective of this study was to compare serum levels of BDNF in patients with PD and control subjects, and to investigate the relationship between BDNF serum levels in PD patients and their motor performance, assessed by using different clinical instruments.
Methods Subjects Demographic and clinical data were collected from 47 PD patients followed at the Movement Disorders Clinic of the University Hospital of the Federal University of Minas Gerais (UFMG) in Belo Horizonte, Brazil. For comparison, 23 healthy controls were recruited. Approval from the local ethics committee was obtained and all participants signed informed consent prior to data collection. For the PD group, inclusion criteria were PD diagnosis and the ability to walk independently. The exclusion criteria were the presence of dementia, delirium, a comorbid neurological or inflammatory disease or a history of neurosurgical procedure. Instruments All participants underwent the Mini-Mental Status Examination (MMSE) and the Beck Depression Inventory (BDI). PD patients also underwent the Unified Parkinson’s
541
Disease Rating Scale (UPDRS), the Modified Hoehn and Yahr Staging Scale (HY), the Modified Schwab and England Activities of Daily Living Scale (S&E), the Berg Balance Scale (BBS), the Timed Up & Go Test (TUG), the Comfortable Gait Speed Test using a 10-meter walk (CGS) and the Six-Minute Walk Test (6MWT). The MMSE was used to assess general cognitive functioning and the recommended adaptations of its scoring for Brazilian elderly were considered [13]. The BDI is the most often used self-rating instrument for depressive symptoms in the clinical setting. According to Silberman et al. and Tumas et al., a cutoff score of 17/18 provides optimal discrimination between depressed and nondepressed Brazilian PD patients [14, 15]. This cutoff score was used to categorize patients into depressed and nondepressed groups. The UPDRS is currently the most widely accepted scale for measuring the different components of PD [16]. It comprises three subscales: UPDRS I—mentation, behavior and mood (range 0–16); UPDRS II—activities of daily living (ADL) (range 0–52) and UPDRS III—motor examination (range 0–108). A score of 176 represents maximal (or total) disability and 0 represents no disability whatsoever. The S&E is widely used to assess any disabilities in performing ADL for people with PD, with 100% representing completely normal functions and 0% representing total helplessness [16]. The HY classifies PD patients into five stages according to the body distribution of symptoms and dependence. Patients in stage I are mildly affected, while in stage V they are bedridden [17]. Other scales were used for a more comprehensive assessment of motor functioning in PD. The BBS is widely used as a balance measure and it was recently validated for PD patients [18]. The TUG measures the time an individual takes to stand up from an armchair, walk along a linear path for 3 m, turn round, walk back towards the chair and sit down, after a ‘‘go’’ oral command [19]. Gait speed is a measure of overall walking performance, but it does not include an endurance component. The 10-meter walk test is a simple, effective and widely used tool to evaluate gait speed [20]. The 6MWT evaluates endurance by measuring the maximum distance an individual can walk within 6 min, and determines the walking capacity [21]. Blood sampling and biochemical measurements Peripheral blood was collected in the morning prior to clinical assessment. Blood samples were centrifuged at 1,000g for 10 min. Next, the serum was removed and stored at -20°C until the BDNF analysis was performed. Blood samples from the control group were processed likewise.
123
542
The concentration of BDNF in the serum of patients and controls was measured according to the procedure supplied by the manufacturer and using sandwich ELISA kits for BDNF (DuoSet, R&D Systems, Minneapolis, MN, USA). All samples were assayed in duplicate. The detection limit for these assays was 10 pg/ml. In brief, the capture antibody (concentration provided by the manufacturer) was diluted in phosphate-buffered saline (PBS), added to each well and left overnight at 4°C. The plate was washed 4 times in PBS with 0.05% Tween 20 (Sigma, St. Louis, MO, USA). The plate was blocked with 1% bovine serum albumin and incubated for 2 h at room temperature before being washed 4 times with PBS and 0.05% Tween 20. The samples and standards were added and the plate incubated overnight at 4°C. After washing the plate, detection antibody (concentration provided by the manufacturer) diluted in PBS was added. The plate was incubated for 2 h at room temperature. After washing the plate, streptavidin (DuoSet R&D Systems, Minneapolis, MN, USA) was added and the plate was incubated for 30 min. Finally, o-phenylenediamine color reagent (Sigma, St. Louis, MO, USA) was added to each well and the reaction was allowed to develop in the dark for 15 min. The reaction was stopped with the addition of 1 M H2SO4 to each well. The absorbance was read on a plate reader at 492 nm wavelengths (Emax, Molecular Devices, Minneapolis, MN, USA). Statistical analysis For comparison of continuous variables, the Student’s t test or the Mann–Whitney U test were used for normally or non-normally distributed data, respectively. The v2 test was used to compare categorical data. Correlation analysis between scales was performed using Pearson’s correlation coefficient or Spearman’s rank correlation coefficient in normally or non-normally distributed data, respectively. Variables that were significantly related to BDNF serum levels in univariate analysis were selected for multiple regression analysis. Indices of goodness of fit of the estimated parameters were calculated after the construction of the regression model. Statistical significance was set at p \ 0.05. The SPSS v15.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.
Results Characteristics of PD patients and control subjects Participants included 47 PD patients (24 men, 23 women) and 23 healthy controls (8 men, 15 women). Mean (±SD) age and MMSE score in control subjects were 61.8 (±10.7) [range: 50–85] years and 25.8 (±3.8) [range: 18–30], while
123
J Neurol (2010) 257:540–545
in PD patients these values were 65.7 (± 8.8) [range: 50– 84] years and 24.4 (± 3.9) [range: 18–30], respectively. Samples did not differ in gender (p = 0.215), age (p = 0.106) or cognitive function as assessed by the MMSE (p = 0.160). The demographic and clinical characteristics of the PD patients are displayed in Table 1. The severity of PD symptoms according to the UPDRS scores was moderate in most participants. The median HY disease staging was 2, compatible with mild-to-moderate disease. The median S&E was 80%, suggesting that most patients were functionally independent. The TUG correlated with the duration of the disease (rs = 0.303, p = 0.039). The BBS, CGS and 6WMT correlated with the duration of the disease (rs = -0.573, p \ 0.001; rs = -0.372, p = 0.010; rs = -0.514, p \ 0.001, respectively), severity of PD symptoms as assessed by UPDRS (rs = -0.847, p \ 0.001; rs = -0.318, p = 0.030; rs = -0.360, p = 0.013, respectively), advanced stage of the disease as classified by the HY (rs = -0.855, p \ 0.001; rs = -0.330, p = 0.023; rs = -0.384, p = 0.008, respectively) and worse level of functional independence on the S&E (rs = -0.836, p \ 0.001; rs = 0.414, p = 0.004; rs = 0.450, p = 0.002, respectively).
Table 1 Demographic and clinical features of Parkinson’s disease patients (n = 47) Variables
Mean ± SD (range)
Age (years)
65.7 ± 8.8 (50–84)
Age of PD onset (years)
58.5 ± 9.7 (40–75)
Disease duration (years)
7.6 ± 4.5 (1–15)
Taking L-dopaa
33
MMSE
24.4 ± 3.9 (18–30)
BDI
15.5 ± 9.9 (0–46)
UPDRS
49.8 ± 29.3 (10–110)
UPDRS I
2.4 ± 2.0 (0–9)
UPDRS II
13.4 ± 8.7 (1–40)
UPDRS III
34.5 ± 22.3 (7–86)
HYb S&Eb
2 (1–4) 80% (40–100%)
BBS
48.9 ± 7.9 (28–56)
TUG
13.8 ± 5.4 (9.5–39.9)
CGS
1.0 ± 0.2 (0.4–1.5)
6MWT
403.9 ± 102.2 (155–570)
SD standard deviation, MMSE Mini-Mental State Examination, BDI Beck Depression Inventory, UPDRS Unified Parkinson’s Disease Rating Scale, HY Hoehn–Yahr Staging Scale, S&E Schwab and England Activities of Daily Living Scale, BBS Berg Balance Scale, TUG Timed Up & Go, CGS Comfortable Gait Speed, 6MWT SixMinute Walk Test a Number of subjects b
Median (range)
J Neurol (2010) 257:540–545
543
Serum levels of BDNF BDNF serum levels of PD patients and controls differed significantly (p = 0.046). Mean (±SD) BDNF level was 4061.6 (±2,374) pg/ml [range: 409.1–8826.7] in PD patients, while in controls it was 5,662 (± 3342.6) pg/ml [range: 626.9–12321.5] (Fig. 1). In PD patients, higher levels of BDNF associated with longer disease duration, more severe degree of impairment caused by the disease as assessed by the UPDRS, especially on subscales II (ADL) and III (motor examination), advanced HY stage and worse performance in ADL, as assessed by the S&E. Higher BDNF levels also correlated with a poor balance on the BBS, as well as with more time spent at the TUG, reduced gait speed and shorter distance covered in the 6MWT (Table 2). BDNF levels did not correlate with BDI scores. Furthermore, there was no difference in serum BDNF levels between PD patients with (n = 14) and without depression (n = 33) (p = 0.762). There was no difference in BDNF serum levels between PD patients treated or not treated with L-dopa (p = 0.461). Multiple regression analyses were conducted to evaluate how well explanatory variables predicted lower serum levels of BDNF. Analyzed variables included: disease duration, UPDRS, HY, S&E, BBS, TUG, CGS and 6MWT scores. Duration of disease, UPDRS, BBS and TUG scores accounted for 50% of the variance of BDNF serum levels in the sample (Table 3).
Table 2 Correlation between serum levels of brain-derived neurotrophic factor (BDNF) and demographic and clinical variables in Parkinson’s disease patients (n = 47) Variables
BDNF Correlation coefficient
p value
Age*
r = 0.133
Age of PD onset*
r = - 0.126
Disease duration*
r = 0.526
MMSE
rs = - 0.172
0.248
BDI
rs = 0.207
0.163
UPDRS I
rs = 0.127
0.394
UPDRS II
rs = 0.458
0.001
UPDRS III
rs = 0.439
0.002
UPDRS Total
rs = 0.479
0.001
HY
rs = 0.355
0.014
S&E
rs = -0.474
0.001
BBS
rs = -0.379
0.009
TUG
rs = 0.430
0.003
rs = -0.450 rs = -0.488
0.001 0.001
CGS 6MWT
0.373 0.397
