Detection of Oligomeric Forms of {Alpha}-Synuclein Protein In Human Plasma As a Potential Biomarker for Parkinson's Disease (original) (raw)
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JAMA Neurology, 2017
IMPORTANCE Parkinson disease (PD) is a highly prevalent and incurable neurodegenerative disease associated with the accumulation of misfolded α-synuclein (αSyn) aggregates. An important problem in this disease is the lack of a sensitive, specific, and noninvasive biochemical diagnosis to help in clinical evaluation, monitoring of disease progression, and early differential diagnosis from related neurodegenerative diseases. OBJECTIVE To develop a novel assay with high sensitivity and specificity to detect small quantities of αSyn aggregates circulating in cerebrospinal fluid (CSF) of patients affected by PD and related synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS The strategy evaluated in this proof-of-concept study uses the protein misfolding cyclic amplification (PMCA) technology that detects minute amounts of misfolded oligomers by taking advantage of their ability to nucleate further aggregation, enabling a very high amplification of the signal. The technology was first adapted with synthetic αSyn oligomers prepared in vitro and used to screen in 2 blinded cohorts of CSF samples from German and Japanese patients with PD (n = 76) and individuals serving as controls affected by other neurologic disorders (n = 65), neurodegenerative diseases (n = 18), and Alzheimer disease (n = 14). The kinetics of αSyn aggregation were measured by αSyn-PMCA in the presence of CSF samples from the participants to detect αSyn oligomeric seeds present in this biological fluid. The assays were conducted from November 15, 2013, to August 28, 2015. MAIN OUTCOMES AND MEASURES Kinetic parameters correlated with disease severity at the time of sample collection, measured by the Hoehn and Yahr scale, with the lowest grade indicating unilateral involvement with minimal or no functional impairment, and the highest grade defining patients with complete confinement to wheelchair or bed. RESULTS Studies with synthetic αSyn aggregates showed that αSyn-PMCA enabled to detect as little as 0.1 pg/mL of αSyn oligomers. The αSyn-PMCA signal was directly proportional to the amount of αSyn oligomers added to the reaction. A blinded study of CSF samples correctly identified patients affected by PD with an overall sensitivity of 88.5% (95% CI, 79.2%-94.6%) and specificity of 96.9% (95% CI, 89.3%-99.6%). The αSyn-PMCA results for different patients correlated with the severity of the clinical symptoms of PD (Japanese cohort: r s = −0.54, P = .006; German cohort: r s = −0.36, P = .02). CONCLUSIONS AND RELEVANCE The findings suggest that detection of αSyn oligomers by αSyn-PMCA in the CSF of patients affected by PD may offer a good opportunity for a sensitive and specific biochemical diagnosis of the disease. Further studies are needed to investigate the usefulness of αSyn-PMCA to monitor disease progression and for preclinical identification of patients who may develop PD.
Movement Disorders, 2019
A BS TRACT: Background: PD diagnosis is based primarily on clinical criteria and can be inaccurate. Biological markers, such as α-synuclein aggregation, that reflect ongoing pathogenic processes may increase diagnosis accuracy and allow disease progression monitoring. Though α-synuclein aggregation assays have been published, reproducibility, standardization, and validation are key challenges for their development as clinical biomarkers. Objective: To cross-validate two α-synuclein seeding aggregation assays developed to detect pathogenic oligomeric α-synuclein species in CSF using samples from the same PD patients and healthy controls from the BioFIND cohort. Methods: CSF samples were tested by two independent laboratories in a blinded fashion. BioFIND features standardized biospecimen collection of clinically typical moderate PD patients and nondisease controls. α-synuclein aggregation was measured by protein misfolding cyclic amplification (Soto lab) and real-time quaking-induced conversion (Green lab). Results were analyzed by an independent statistician. Results: Measuring 105 PD and 79 healthy control CSF samples, these assays showed 92% concordance. The areas under the curve from receiver operating characteristic curve analysis for the diagnosis of PD versus healthy controls were 0.93 for protein misfolding cyclic amplification, 0.89 for real-time quaking-induced conversion, and 0.95 when considering only concordant assay results. Clinical characteristics of false-positive and-negative subjects were not different from true-negative and-positive subjects, respectively. Conclusions: These α-synuclein seeding aggregation assays are reliable and reproducible for PD diagnosis. Assay parameters did not correlate with clinical parameters, including disease severity or duration. This assay is highly accurate for PD diagnosis and may impact clinical practice and clinical trials.
The FASEB Journal, 2006
To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). ␣-Synuclein (␣-syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding ␣-syn in familial cases with early-onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of ␣-syn into insoluble aggregates. We recently reported the presence of ␣-syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether ␣-syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric "soluble aggregates" of ␣-syn. Using this ELISA, we report the presence of significantly elevated
The FASEB journal, 2006
To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analysis of blood or cerebrospinal fluid (CSF). ␣-Synuclein (␣-syn) protein has been linked to the pathogenesis of PD with the discovery of mutations in the gene encoding ␣-syn in familial cases with early-onset PD. Lewy bodies and Lewy neurites, which constitute the main pathological features in the brains of patients with sporadic PD and dementia with Lewy bodies, are formed by the conversion of soluble monomers of ␣-syn into insoluble aggregates. We recently reported the presence of ␣-syn in normal human blood plasma and in postmortem CSF. Here, we investigated whether ␣-syn can be used as a biomarker for PD. We have developed a novel ELISA method that detects only oligomeric "soluble aggregates" of ␣-syn. Using this ELISA, we report the presence of significantly elevated
Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical motor features associated. Pathologically, it is characterized by the intracellular accumulation of alpha-synuclein (aSyn) in Lewy bodies and Lewy neurites. Currently, there are no established biochemical markers for diagnosing or for following disease progression, a major limitation for the clinical practice. Posttranslational modifications (PTMs) in aSyn have been identified and implicated on its pathobiology. Since aSyn is abundant in blood erythrocytes, we aimed to evaluate whether PTMs of aSyn in the blood might hold value as a biomarker for PD. We examined 58 patients with PD and 30 healthy age-matched individuals. We found that the levels of Y125 phosphorylated, Y39 nitrated, and glycated aSyn were increased in PD, while those of SUMO were reduced. A combinatory analysis of the levels of these PTMs resulted in an increased sensitivity, with an area under curve (AUC) of 0.843 for PD versus healthy controls, and correlated with disease severity and duration. We conclude that the levels of these selected PTMs hold strong potential as biochemical markers for PD. Ultimately, our findings might facilitate the monitoring of disease progression in clinical trials, opening the possibility for developing more effective therapies against PD. Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder affecting 1–2% of people over the age of 65 years 1. The diagnosis of PD is currently based on typical clinical motor features, such as bradykinesia, resting tremor, or rigidity 2. The response to levodopa treatment represents an important factor for diagnosis, since PD patients usually present a good response to this medication 3. Different clinical scales are routinely used to classify and stage the disease. The Movement Disorder Society (MDS) recently revised the Unified PD Rating Scale (MDS-UPDRS), which is subdivided into four components 4. Parts I and II are associated to the non-motor and motor experiences of everyday, while part III is reserved to the motor evaluation and finally Part IV is related to motor complications 5. The Hoehn and Yahr (HY) scale is divided into 5 stages that reflect motor progression of the disease 6. However, misdiagnosis of PD is rather common 7,8 , affecting therapeutics and complicating patient selection for clinical trials. Thus, it is urgent to identify novel and objective biomarkers to unequivocally diagnose and follow disease progression. Pathologically, PD is characterized by the loss of nigrostriatal dopaminergic neurons and the accumulation of neuronal cytoplasmic inclusions known as Lewy bodies (LBs) or Lewy neurites, which are primarily composed of the protein alpha-synuclein (aSyn) 9. The majority of PD cases are sporadic, however several genes have been associated with familial cases 10. Mutations and multiplications in the gene encoding for aSyn are also associated with familial forms of PD 11. Interestingly, in both sporadic and familial forms of PD the progression and severity of the disease correlate with the distribution of aSyn inclusions 12. For reasons we still do not fully understand, aSyn is prone to misfolding and self-association into high molecular weight species, ultimately forming LBs 13. Posttranslational modifications (PTMs) have a direct impact in the aggregation and toxicity of aSyn. The protein undergoes several PTMs such as acetylation and glycation, as we recently described 14,15 , as well as oxidation, phosphorylation, nitration, sumoylation, and ubiquitination 16–18. We showed that while acetylation protects from aSyn aggregation and toxicity 14 , glycation exacerbates oligomeriza-tion and cytotoxicity, inducing dopaminergic neuronal death 15. Phosphorylation has been extensively studied PTM since most aSyn found in LBs is phosphorylated on serine 129 19,20. However, the effects of S129 phosphoryl-ation are still controversial 21–25. Other modifications, such as nitration 26 and oxidation 27 enhance, while sumoyla-tion 28 prevents/reduces the aggregation of aSyn. Nevertheless, although PTMs may play important roles on aSyn biology, our understanding of the precise effects of PTMs on aSyn is still limited. aSyn is present in various body fluids such as cerebrospinal fluid (CSF) and blood 29,30. Thus, the presence of this protein in easily-accessible body fluids opens the possibility of investigating PTMs in aSyn. Taking advantage of the thermo-stability of aSyn 31 , we detected PTMs in aSyn extracted from brain tissue or from cultured cells 14,15,32. Although the pool of aSyn in the blood may differ from that in the brain or CSF, we hypothesized that, given the central role this protein plays in PD, it might report on disease-related alterations that may be used as bio-markers for PD. This is in line with the observation that aSyn can be secreted from neuronal cells 33 , and that it is phosphorylated on serine 129 in the serum of PD patients 34. In this study, we identified a specific pattern of aSyn PTMs that discriminated PD patients from controls individuals. As these PTMs correlate with disease severity and duration, we propose that these modifications may possibly be used as biochemical markers for PD. Results Patient population and demographics. We recruited a group of 88 individuals, divided into 3 subgroups: 28 diagnosed with PD for 2–4 years, 30 diagnosed for ≥10 years, and 30 controls. Blood samples were processed by the same personnel. The PD group diagnosed with the disease for 2–4 years presented a male to female (M:F) ratio of 1: 1, a mean age at onset of 69.0 ± 10.3 years, a mean disease duration of 3.1 ± 0.7 years, a MDS-UPDRS III of 38.4 ± 10.8 and a HY of 1.6 ± 0.8. The PD group diagnosed for ≥10 years presented a M:F ratio of 1: 1, a mean age of onset of 67.2 ± 7.6 years, a mean disease duration of 17.7 ± 4.3, a MDS-UPDRS III of 60.0 ± 19.9 and a HY of 3.0 ± 1.1. The mean age of controls was 67.2 ± 7.6 years. Male to female ratio in the control group was 1:2 (Table 1 and Fig. S1). aSyn purification and enrichment. To analyse PTMs of aSyn in blood, we first enriched the aSyn content from erythrocyte lysates taking advantage of the thermo-stability of aSyn, as previously described 32. Briefly, as described in Fig. 1a–d, by heating protein samples, non-thermo-stable proteins precipitated, while aSyn remained in the soluble fraction, enabling its detection (Fig. 1b–d). As haemoglobin is the major protein component of erythrocytes lysates (90%), we depleted this protein from thermo-enriched erythrocyte lysates using HemoVoid, a silica-based protein enrichment matrix that removes haemoglobin from lysates, enabling the additional concentration of other proteins of lower abundance 35. We confirmed the aSyn enrichment by immunoblotting (SDS-PAGE and dot-blot) (Fig. 1c,d). Thus, for all subsequent analysis, the erythrocyte fractions were thermo-enriched and haemoglobin depleted. Although the amount of haemoglobin is severely reduced in thermo-enriched-haemoglobin-depleted (TE-HD) extracts, and a band at 15 kDa is evident, other higher molecular weight bands are also observed (Fig. 1b).
α-Synuclein and DJ-1 as potential biological fluid biomarkers for Parkinson's Disease
International journal of molecular sciences, 2010
Parkinson's disease (PD) is the most common form of movement disorder and affects approximately 4% of the population aged over 80 years old. Currently, PD cannot be prevented or cured, and no single diagnostic biomarkers are available. Notably, recent studies suggest that two familial PD-linked molecules, α-synuclein and DJ-1, are present in cerebrospinal fluid (CSF) and that their levels may be altered during the progression of PD. In this regard, sensitive and accurate methods for evaluation of α-synuclein and DJ-1 levels in the CSF and blood have been developed, and the results suggest that the levels of both molecules are significantly decreased in the CSF in patients with PD compared with age-matched controls. Furthermore, specific detection and quantification of neurotoxic oligometric forms of α-synuclein in the blood using enzyme-linked immunosorbent assays might be expected as potential peripheral biomarkers for PD, although further validation is required. Currently, nei...
DJ-1 and -synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease
Brain, 2010
Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson's disease. Both DJ-1 and a-synuclein, two proteins critically involved in Parkinson's disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and a-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and a-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and a-synuclein from 117 patients with Parkinson's disease, 132 healthy individuals and 50 patients with Alzheimer's disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and a-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and a-synuclein levels were decreased in Parkinson's patients versus controls or Alzheimer's patients when blood contamination was controlled for. In the population aged 565 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and a-synuclein, respectively, the sensitivity and specificity for patients with Parkinson's disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for a-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or a-synuclein and the severity of Parkinson's disease. Taken together, this represents the largest scale study for DJ-1 or a-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and a-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinson's disease, if variables such as blood contamination and age are taken into consideration. Abbreviations: ELISA = enzyme-linked immunosorbent assay; MMSE = mini-mental state exam; ROC = receiver operating characteristic
ACS chemical neuroscience, 2017
Autoantibodies to Parkinson's disease (PD) amyloidogenic protein, α-synuclein, were recognized as a prospective biomarker for early disease diagnostics, yet there is inconsistency in previous reports, potentially related to PD status. Therefore, plasma and cerebrospinal fluid (CSF) of the cross-sectional cohort of 60 individuals, including recently diagnosed PD patients with mild and moderate PD and age-matched controls, were examined by enzyme-linked immunosorbent assay (ELISA). Nonparametric statistics was used for data analysis. We found significantly elevated levels of α-synuclein autoantibodies in both plasma and CSF in mild PD compared to controls, followed by some decrease in moderate PD. Receiver operating characteristic and effect size analyses confirmed the diagnostic power of α-synuclein antibodies in both plasma and CSF. For the first time, we showed the correlation between plasma and CSF α-synuclein antibody levels for mild, moderate, and combined PD groups. This in...
2014
The quantification of a-synuclein (aSyn) in cerebrospinal fluid (CSF) has been proposed as a diagnostic biomarker for Parkinson's disease and other aSyn-related diseases, such as multiple system atrophy and dementia with Lewy bodies. Most studies show decreased levels of aSyn in diseased CSF samples compared to control samples, but discrepant findings and overlapping values have been a major limitation for the use of CSF aSyn as a biomarker. This review addresses the current knowledge and investigates whether CSF aSyn is an ideal biomarker that can detect fundamental neuropathology features. It will also discuss whether CSF aSyn has been validated in neuropathologically confirmed cases, whether it shows a diagnostic sensitivity and whether it has a specificity above 80%. The review of current literature will also determine if sampling CSF aSyn is reliable, reproducible, noninvasive, simple to perform, inexpensive, and whether it has been investigated by at least two independent studies. CSF aSyn appears to meet most of these criteria, which have been proposed for ideal biomarkers, but further validation of this and other markers is needed to best introduce a panel of biomarkers in the early and differential diagnosis of Parkinson's disease.
PLoS ONE, 2011
Background: Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson's (PD) and Alzheimer's diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer's disease and potentially other neurodegeneration ailments.