Dataset Information

In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

ABSTRACT: The aggregation of alpha-synuclein (?Syn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of ?Syn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between ?Syn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human ?Syn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human ?Syn, a PLA signal indicating close proximity between ?Syn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human ?Syn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of ?Syn and the SNARE proteins in primary neuronal cultures.

SUBMITTER: Almandoz-Gil L

PROVIDER: S-EPMC5874290 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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<i>In Situ</i> Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

Almandoz-Gil Leire L Persson Emma E Lindström Veronica V Ingelsson Martin M Erlandsson Anna A Bergström Joakim J

Frontiers in neurology 20180322

The aggregation of alpha-synuclein (αSyn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of αSyn is to promote the soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was ...[more]

PMID: 29623065

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Project description:BACKGROUND: Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of alpha-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, alpha-synuclein adopts a helical conformation that mediates specific interactions with other proteins. RESULTS: To investigate the role of the helical domain in transport and localization of alpha-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and alpha-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of alpha-synuclein (exons 2, 3, and 4) partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged alpha-synuclein migrated at a rate of 1.85 +/- 0.09 mum/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Delta234GFP) did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. CONCLUSIONS: These results support the hypothesis that presynaptic localization involves a mechanism that requires helical conformation and lipid binding. Conversely, the rate of axonal transport is not determined by lipid affinity and is not sufficient to account for differences in presynaptic localization of alpha-synuclein-eGFP variants.

Dataset Information

In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

Publications

<i>In Situ</i> Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

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