Dataset Information

Identification of proteins interacting with extracellularly applied α-syn assemblies, Condition 3 - controls (5/8)

ABSTRACT: Fibrillar α-Synuclein (α-Syn) is the principal component of Lewy bodies which are evident in individuals affected by Parkinson disease (PD). This neuropathologic form of α-Syn contributes to PD progression and propagation of exogeneous α-Syn between neurons has been demonstrated. In order to identify proteins interacting with extracellularly applied α-syn assemblies (either oligomeric or fibrillar α -syn) and identify in particular plasma membrane proteins exposed extracellularly, we exposed pure-neuronal cultures to oligomeric and fibrillar α-syn for 10 min, pulled down the complex, and identified the associated proteins using a proteomic-based approach. Using pull-down of whole cell lysates and MS, we have identified proteins interacting with extracellularly applied α-syn in three different conditions. Each condition consisted in three experimental replicates of cells exposed 10 min to α-syn assemblies and the non-treated cells used as controls.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain, Astrocyte

DISEASE(S): Parkinson's Disease

SUBMITTER: Virginie Redeker

LAB HEAD: Virginie Redeker; Ronald Melki

PROVIDER: PXD002260 | Pride | 2015-09-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Condition 3- Astrocytes 10 min Ctrl1.pride.mztab.gz	Mztab
	Condition 3- Astrocytes 10 min Ctrl2.pride.mztab.gz	Mztab
	Condition 3- Astrocytes 10 min Ctrl3.pride.mztab.gz	Mztab
	Condition3-Astrocytes10minCtrl1.dat	Other
	Condition3-Astrocytes10minCtrl1.mzid.gz	Mzid

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Publications

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

Shrivastava Amulya Nidhi AN Redeker Virginie V Fritz Nicolas N Pieri Laura L Almeida Leandro G LG Spolidoro Maria M Liebmann Thomas T Bousset Luc L Renner Marianne M Léna Clément C Aperia Anita A Melki Ronald R Triller Antoine A

The EMBO journal 20150831 19

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, ...[more]

PMID: 26323479

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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.