Dataset Information

Identification of Alpha-Synuclein Interacting Synaptosomal Proteins

ABSTRACT: α-Synuclein is an abundant presynaptic protein that when aggregated and dyslocated from the synapse is associated to Parkinson’s disease and dementia. The normal presynaptic function of α-synuclein is unclear as well as the disease triggering mechanism. In order to extend the knowledge of the α-synuclein interactome during normal function and disease, we have used porcine brain synaptosomes as a source of ligands and purified α-synuclein monomers or oligomers as bait in co-immunoprecipitation experiments. The isolated binding synaptosomal proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using the freely-available Windows client application, Skyline Targeted Proteomic Environment. To compare proteins binding to a-synuclein monomer with proteins binding to a-synuclein oligomer, quantifications were log transformed, normalized to buffer-background, and compared by students t-test. Furthermore, to specify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. 10 α-synuclein preferential monomer binding proteins were identified and among those, we successfully validated Abl interactor 1, and myelin proteolipid protein. 76 α-synuclein preferential oligomer binding proteins were found, including glutamate decarboxylase 2, synapsin 1, and glial fibrillary acidic protein, which were positively validated. We identified 92 proteins binding to α-synuclein, for which we were not able to detect any conformational preferences among. This study presents a catalog of proteins interacting with α-synuclein in its non-aggregated and aggregated oligomeric state, which can be used to investigate the normal and pathological roles of α-synuclein.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Brain

DISEASE(S): Parkinson's Disease

SUBMITTER: Min Shi

LAB HEAD: Poul Henning Jensen

PROVIDER: PXD001462 | Pride | 2015-02-09

REPOSITORIES: Pride

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Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.

Betzer Cristine C Movius A James AJ Shi Min M Gai Wei-Ping WP Zhang Jing J Jensen Poul Henning PH

PloS one 20150206 2

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson's disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function asso ...[more]

PMID: 25659148

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Project description:Abnormal accumulation of aggregated proteins and sustained microglial activation are important contributors of neurodegenerative process in neurological diseases. Recent studies have shown that aggregation-prone proteins, such as a-synuclein, the protein implicated in Parkinson’s disease (PD), are released from neuronal cells and thus present in the extracellular fluid, pointing to the possible paracrine effects of these proteins on microglial immune responses. However, the mechanism underlying the disease-associated microglial activation and the role of neuronal proteins in this process remain unknown. Here, we show that extracellular a-synuclein released from neuronal cells is an endogenous ligand of toll-like receptor 2 (TLR2) and activates microglia, which in turn induces neurodegeneration. Interaction between neuron-released a-synuclein and TLR2 and subsequent activation of the TLR2 signaling were demonstrated comprehensively by using computational modeling of signaling network and by the experimental validation in TLR2-deficient microglia both in vitro and in vivo. In contrast to the neuron-released a-synuclein, recombinant a-synuclein proteins, including monomer, oligomer, fibril, or nitrated forms, were not able to interact or activate TLR2, suggesting that neuronal cells have a mechanism of enriching specific forms of a-synuclein capable of activating TLR2 during the process of releasing this protein. Taken together, the results suggest that both neuron-released extracellular a-synuclein and TLR2 might be novel therapeutic targets for modifying neuroinflammation in PD and related neurodegenerative diseases. We collected culture media from differentiated SH-SY5Y cells overexpressing either human a-synuclein (alpha-SCM) or beta-galactosidase (LZCM) and treat these media to primary rat microglia at the concentration of a-synuclein of 1.1M. Transcriptome analyses with microglial cells treated with either aSCM or LZCM at two different time points, 6 h and 24 h.

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