CSF markers of neurodegeneration in Parkinson’s disease
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- Přikrylová Vranová, H., Mareš, J., Nevrlý, M. et al. J Neural Transm (2010) 117: 1177. doi:10.1007/s00702-010-0462-z
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Parkinson’s disease (PD) is a chronic, progressive, neurodegenerative disease with a multifactorial etiology. Protein accumulation is speculated by some to play a prominent role in the pathogenesis of PD. The severity of neurodegeneration should correlate with cerebrospinal fluid (CSF) levels of these neurodegenerative markers (NDMs). The aims of the study were to assess the CSF levels of tau protein, beta-amyloid (1–42), cystatin C, and clusterin in patients suffering from PD and in a control group, to compare the CSF levels between the two groups and to correlate them to PD duration. NDMs in the CSF were assessed in 32 patients suffering from PD and in a control group (CG) of 30 patients. The following statistically significant differences in the CSF were found: higher tau protein (p = 0.045) and clusterin levels (p = 0.004) in PD patients versus CG; higher tau protein levels (p = 0.033), tau protein/beta-amyloid (1–42) ratio (p = 0.011), and clusterin (p = 0.044) in patients suffering from PD for <2 years versus patients suffering PD for more than 2 years. No differences between beta-amyloid (1–42) and cystatin C CSF levels were found in the CG and PD patients groups. Significantly higher tau protein and clusterin CSF levels in the group of PD patients with disease duration of <2 years probably reflect the fact that most neurodegenerative changes in PD patients occur in the initial stage of disease.
KeywordsParkinson’s diseaseTau proteinClusterinCSFNeurodegeneration
Parkinson’s disease (PD) is a chronic, progressive, neurodegenerative disease with a multifactorial etiology (Marras and Lang 2008; Weintraub et al. 2008). Several mechanisms have been implicated as crucial to PD pathogenesis: oxidative stress (Alam et al. 1997), mitochondrial dysfunction (Schapira 2008), protein aggregation and misfolding (Cookson and van der Brug 2008), inflammation, excitotoxicity, apoptosis and other cell death pathways, and loss of trophic support. No one mechanism appears to be primary in all cases of PD, and these pathogenic mechanisms likely act synergistically through complex interactions to promote neurodegeneration (Yacoubian and Standaert 2009). The pathologic hallmark of PD is degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), resulting in depletion of striatal dopamine (McNaught and Olanow 2006). Some surviving neurons contain eosinophilic intracytoplasmic inclusions, or Lewy bodies, which are in part composed of numerous proteins. Protein accumulation is speculated by some to play a prominent role in the pathogenesis of PD, and the appearance of proteins in Lewy bodies tends to support this notion. The neurodegenerative process in PD is not limited to the SNc, and neuronal loss with Lewy body formation also occurs in other brain regions, which may account for both motor and nonmotor features of the disease (Jenner and Olanow 2006; Braak et al. 2003). An understanding of the mechanisms underlying the development and progression of PD pathology is critical for the development of neuroprotective therapies. Currently, the therapy of PD is rather symptomatic, and its neuroprotective effect is speculative. Moreover, timing of the therapy onset remains controversial. Demonstration of neurodegeneration in the early stage of the disease should lead to consideration of timely therapy with neuroprotective effect.
Neurodegeneration is known to be related to the presence of certain biochemical markers in the cerebrospinal fluid (CSF), tau protein and beta-amyloid (1–42) as markers of protein dysfunction, cystatin C as amyloidogenic protein and clusterin as preventive cell-death protein (Bibl et al. 2006; Mareš et al. 2003). Therefore, the intensity of the neurodegenerative process is thought to be related to particular concentrations of specific biochemical markers (tau protein, beta-amyloid (1–42), cystatin C, clusterin) in the CSF (Vranová et al. 2008, 2009). The presented study aimed to document the neurodegenerative process using determination of the concentrations of these markers in the CSF of both PD patients and control subjects. In addition, this study aimed to seek potential correlation between these levels and the duration of the disease in PD patients.
Patients and methods
The study was approved by the institutional ethics committee and all patients signed an informed consent.
A total of 32 patients suffering from PD without signs of dementia were examined: 10 females, 22 males, aged 37–77 years, mean age 59.9 ± 11.26 years, mean age at onset 55.2 ± 12.77, mean duration of disease 4.4 ± 3.6 years. Twenty patients were treated with l-DOPA of mean dose 492.3 ± 137.2 mg daily, mean duration of treatment with l-DOPA 3.92 ± 3.4 years.
The control group comprised 30 patients: 13 females 17 males, aged 33–79, mean age 58.8 ± 10.22 years. Of those, 12 patients had vertebrogenic disease, 9 patients had psychogenic disease, 5 had migraine, 2 patients had tension headache, and 2 patients had diabetic neuropathy. Parkinson’s disease was diagnosed using the United Kingdom Parkinson’s Disease Society Brain Bank criteria (Hughes et al. 1992). Cognitive deficit was excluded by psychological assessment involving the Mini-Mental Status Examination (MMSE). In all patients, CSF levels of tau protein, beta-amyloid (1–42), cystatin C, and clusterin were measured.
Laboratory analysis and CSF sampling technique
The CSF samples were obtained by lumbar puncture performed in the morning using a 20-gauge atraumatic spinal needle under the usual sterile conditions, with the subjects sitting and the needle being inserted between lumbar vertebrae L4/L5. During each puncture, 10 ml of CSF was collected in a sterile polypropylene tube and immediately frozen at −80°C until analysis. Within 10 weeks of freezing, in all subjects in the series, the concentrations of total tau protein (ELISA, Biosource, Great Britain), beta-amyloid (1–42) (ELISA, Innogenetics, Belgium), cystatin C (ELISA, Biovendor, Czech Republic), and clusterin (ELISA, Biovendor, Czech Republic) were measured. Analytical characteristics of all ELISA methods when verifying the manufacturers’ data were satisfactory (both repeatability and reproducibility <9%). At the same time, the CSF tau protein/beta-amyloid (1–42) ratio was calculated in all patients (Stejskal et al. 2005).
Division of the group into subgroups
To evaluate the studied parameters, the results were compared between the PD patient group and the control group and then between PD patient subgroups.
We would like to correlate NMD levels with disease duration, but it was impossible due to small patient sample size. Our tertiary Movement Disorders center usually gets referred either patients with suspicion for PD or patients with advanced stage of PD. Therefore, our PD group spontaneously divided into two subgroups with duration of ≤2 and >2 years, and the values were compared between these two groups.
Medcalc (Belgium) software was used for statistical analysis. To determine differences between the PD patient group and the control group as well as those in the subgroups of patients with PD, the chi-square test was used for categorical variables. Among continuous variables, Student’s t test was used for age with presumed normal distribution and non-parametric tests for all other assessments. Mann–Whitney test was used for comparing the groups, whereas the possible dependence of values of metric parameters on age was assessed by the non-parametric Spearman’s correlation analysis. The p value <0.05 was considered to be statistically significant. The reason for using nonparametric tests for data analysis was the abnormal distribution of parameters (the Shapiro–Wilk test for normality), namely the presence of extreme values in the data.
Age, mean ± SD (years)
59.9 ± 11.26
58.8 ± 10.22
The presented study showed significantly higher levels of CSF tau protein and clusterin in the PD patients when compared with the control group. At the same time, significantly higher levels of CSF tau protein and clusterin were found in patients with <2 years since the first symptoms had manifested.
Tau protein, under normal conditions, contributes to the integrity of the cytoskeleton. Its excessive phosphorylation, as in the case of numerous neurodegenerative diseases including PD, results in impaired cell integrity, loss of its physiological function, and even cell death. Inclusion of tau protein is found with alpha-synuclein as a part of Lewy bodies in patients with PD (Arima et al. 1999; Esposito et al. 2007). Elevation of total tau protein CSF levels is considered a marker of neurodegeneration. In presented study, the presence of higher tau protein levels is in accordance with the results of previous studies concerning its contribution to the development of the disease, but does not correlate with an earlier study carried out in individuals suffering from PD without signs of dementia (Molina et al. 1997).
Clusterin, a heterodimeric glycoprotein, has been described to attribute to many cellular physiologic functions, including cell–cell interactions, complement inhibition, lipid transportation, cell survival, and apoptosis. Clusterin, also called apoprotein J, is induced under cytotoxic conditions to protect cells from cytotoxic stress (Pucci et al. 2008). It has been reported that clusterin is associated with many degenerative diseases, such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) (Sasaki et al. 2002). Higher clusterin levels in PD patients bear witness to presence of cell-death protective mechanism during neurodegeneration.
At the same time, significantly higher levels of CSF tau protein and clusterin were found in patients with <2 years of disease duration since the first symptoms had manifested. This led to the hypothesis that the highest degree of degeneration (i.e. the most intensive neurodegenerative process) is likely to occur in the initial stage, i.e., when the severity of pathological changes in the brain exceed the threshold of clinical manifestation.
Earlier studies showed higher levels of tau protein in patients with PD with dementia than in those with PD without signs of dementia (Mollenhauer et al. 2006). Therefore, the studied PD patients should be subsequently clinically monitored for cognitive deficit to see whether increased levels of tau protein in the CSF may serve as a potential marker of the later development of dementia (Fagan et al. 2007).
Nevertheless, the study has several limitations. The first limitation is the small size of the PD patient group. The other possible limitation (fairly rather improbable) of the study is that an unspecified neurodegenerative process, not manifested clinically at the time of CSF collection, might have been underway in the control subjects. Likewise in the patients, we do not know the exact timing and evolution of the underlying neurodegenerative process; so our timing is related to symptom onset.
Determining the levels of tau protein in the CSF could be regarded as a potential laboratory marker of the presence of neurodegeneration in PD patients. Higher clusterin CSF levels in PD patients demonstrate that during the neurodegeneration not only pro-apoptotic but also anti-apoptotic mechanisms are presented. Higher clusterin and tau CSF levels in PD patients with <2 years of disease duration also support the hypothesis that the highest rate of degeneration is likely to occur in the early stage of disease.
Conflict of interest
The authors declare that they have no conflict of interest.