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Development of a novel method for the quantification of tyrosine 39 phosphorylated ?- and ?-synuclein in human cerebrospinal fluid.

ABSTRACT: Background:Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 (Y39) of ?-synuclein (?-syn) (pY39 ?-syn), which contributes to the death of dopaminergic neurons. Because pY39 ?-syn may be pathogenic, monitoring pY39 ?-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 ?-syn can be detected in the cerebrospinal fluid (CSF) of PD patients by targeted mass spectrometry. Methods:Here, we report a two-step enrichment method in which phosphotyrosine peptides were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium dioxide (TiO2) beads to detect EGVLpYVGSK sequence derived from tyrosine 39 region of ?- and ?-synuclein (??-syn). Accurate quantification was achieved by adding a synthetic heavy version of pY39 ??-syn peptide before enzymatic digestion. Results:Using the developed enrichment methods and optimized parallel reaction monitoring (PRM) assays, we detected pY39 ??-syn peptide in human CSF and demonstrated that the ratio of pY39 ??-syn to Y39 ??-syn was significantly increased in the CSF of patients with PD. Conclusions:We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.

SUBMITTER: Na CH

PROVIDER: S-EPMC7197159 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of a novel method for the quantification of tyrosine 39 phosphorylated α- and β-synuclein in human cerebrospinal fluid.

Na Chan Hyun CH Sathe Gajanan G Rosenthal Liana S LS Moghekar Abhay R AR Dawson Valina L VL Dawson Ted M TM Pandey Akhilesh A

Clinical proteomics 20200504

<h4>Background</h4>Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation ...[more]

PMID: 32390785

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Project description:Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder caused by dopaminergic neuronal deaths. For a proper treatment of PD, biomarkers that would enable us to monitor disease progression or responses to a disease-modifying agent, or that can be used as a presymptomatic marker are critical. In addition, a biomarker that could be used to distinguish between the various diseases that manifest with PD-like symptoms would greatly enhance patient care and management of future clinical trials. Previous research indicates that c-Abl is overactive in PD patients resulting in an increased phosphorylation of the tyrosine 39 residue of alpha-synuclein (a-syn) (pY39 a-syn) and eventually dopaminergic neuronal death via a-syn aggregation. For this reason, monitoring pY39 a-syn will enable us to diagnose presymptomatic PD, monitoring disease progression and monitoring the response disease-modifying agents. We hypothesized that this increased phosphorylation on Y39 of a-syn (pY39 a-syn) will be reflected in the cerebrospinal fluid (CSF) of PD patients and monitoring pY39 a-syn level in CSF will enable us to monitor pY39 a-syn level in the brain. Mass spectrometry has been widely used for the detection of phosphorylated peptides using parallel reaction monitoring (PRM) and we utilized the PRM mass spectrometry for the detection of pY39 a-syn. However, pY39 a-syn was not detected even after immunoprecipitation of a-syn, phosphopeptide enrichment or phosphotyrosine peptide enrichment from a practical volume. For this reason, we have developed a two-step enrichment method in which pY39 a-syn was first enriched with an anti-phosphotyrosine antibody followed by a further enrichment of phosphopeptides with titanium oxide (TiO2) beads to remove non-phosphopeptides. Accurate quantification was obtained by including a synthetic pY39 a-syn peptide labeled with heavy lysine that was added before an enzyme digestion. To detect pY39 α-syn peptide in CSF lower than 10 femtomolar concentration, experimental parameters were optimized. Comparison of pY39 a-syn to total a-syn in CSF from PD patients and controls reveals that pY39 a-syn levels are significantly elevated in the CSF of PD patient versus controls. This optimized two-step enrichment PRM detection method may be of value as a biomarker to monitor c-Abl activation in PD patients for future diagnostic studies as well as a pharmacodynamic marker for c-Abl therapeutic trials in PD. Furthermore, this developed method can be applicable to quantifying other phosphotyrosine peptides with low abundance in biological samples.

Project description:?-Synuclein (?Syn) is a major constituent of proteinaceous aggregates in neurodegenerative diseases such as Parkinson's disease (PD) and a potential biomarker candidate for diagnosis and treatment effects. However, studies about ?Syn in cerebrospinal fluid (CSF) in diseases are inconsistent and mainly based on immunological assays. Quantitative information about ?-synuclein (?Syn) and ?-synuclein (?Syn) in CSF is not available.Here, we present an alternative method for the simultaneous quantification of ?Syn, ?Syn and ?Syn in CSF by multiple reaction monitoring (MRM) with a high sequence coverage (70%) of ?Syn to validate previous, ELISA-based results and characterize synucleins in CSF in more detail.The MRM has high sensitivity in the low pg/ml range (3-30pg/ml full-length ?Syn) using 200 ?l CSF. A high portion of CSF ?Syn is present in the N-terminally acetylated form and the concentration of unmodified peptides in the nonamyloid component region is about 40% lower than in the N-terminal region. Synuclein concentrations show a high correlation with each other in CSF (r>0.80) and in contrast to ?Syn and ?Syn, ?Syn is not affected by blood contamination. CSF ?Syn, ?Syn and ?Syn concentrations were increased in Alzheimer's and Creutzfeldt-Jakob disease but not altered in PD, PD dementia (PDD), Lewy body dementia and atypical parkinsonian syndromes. The ratio ?Syn/?Syn was increased in PDD (1.49 ± 0.38, p < 0.05) compared with PD (1.11 ± 0.26) and controls (1.15 ± 0.28). ?Syn shows a high correlation with CSF tau concentrations (r = 0.86, p < 0.0001, n = 125).In conclusion, we could not confirm previous observations of reduced ?Syn in PD and our results indicate that CSF synuclein concentrations are rather general markers of synaptic degeneration than specific for synucleinopathies. ?syn is an attractive biomarker candidate that might be used as an alternative to or in combination with tau in AD and CJD diagnosis and in combination with ?Syn it is a biomarker candidate for PDD.

Project description:BackgroundThe objective of this study was to investigate the discriminating value of a range of CSF α-synuclein species for dementia with Lewy bodies compared with Alzheimer's disease, PD, and cognitively normal controls.MethodsWe applied our recently published enzyme-linked immunosorbent assays to measure the CSF levels of total α-synuclein, oligomeric α-synuclein, and phosphorylated α-synuclein in dementia with Lewy bodies (n = 42), Alzheimer's disease (n = 39), PD (n = 46), and controls (n = 78). General linear models corrected for age and sex were performed to assess differences in α-synuclein levels between groups. We used backward-elimination logistic regression analysis to investigate the combined discriminating value of the different CSF α-synuclein species and Alzheimer's disease biomarkers.ResultsCSF levels of total α-synuclein were lower in dementia with Lewy bodies and PD compared with Alzheimer's disease as well as controls (P < 0.001). In contrast, CSF levels of oligomeric α-synuclein were higher in dementia with Lewy bodies and PD compared with Alzheimer's disease (P < 0.05) and controls (P < 0.001). No group differences were found for phosphorylated α-synuclein. In dementia with Lewy bodies and PD, CSF total α-synuclein levels positively correlated with tau and phosphorylated tau (both r > 0.40, P < 0.01), but not with amyloid-β1-42 . The optimal combination to differentiate dementia with Lewy bodies from controls consisted of amyloid-β1-42 , tau, total α-synuclein, oligomeric α-synuclein, age, and sex (AUC, 0.90). To differentiate dementia with Lewy bodies from Alzheimer's disease, the combination of tau and oligomeric α-synuclein resulted in an AUC of 0.83. CSF α-synuclein species do not contribute to the differentiation of dementia with Lewy bodies from PD.ConclusionsCSF α-synuclein species could be useful as part of a biomarker panel for dementia with Lewy bodies. Evaluating both oligomeric α-synuclein and total α-synuclein in CSF helps in the diagnosis of dementia with Lewy bodies. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Dataset Information

Development of a novel method for the quantification of tyrosine 39 phosphorylated ?- and ?-synuclein in human cerebrospinal fluid.

Publications

Development of a novel method for the quantification of tyrosine 39 phosphorylated α- and β-synuclein in human cerebrospinal fluid.

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