Abstract
BackgroundAlpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of α-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, α-synuclein adopts a helical conformation that mediates specific interactions with other proteins.ResultsTo investigate the role of the helical domain in transport and localization of α-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 α-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 α-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 α-synuclein migrated at a rate of 1.85 ± 0.09 μm/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 (Δ234GFP) 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.ConclusionsThese 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 α-synuclein-eGFP variants.
Highlights
Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis
We found that deletions of single exons within the lipid-binding N-terminal domain of a-syn or the Parkinson’s disease (PD)-association mutation A30P partially disrupted the localization of a-syn at presynaptic terminals, resulting in increased diffuse labeling of axons
As observed previously for wild-type a-syn [27], ASGFP stably localizes to presynaptic terminals of primary hippocampal neurons by approximately 14-21 days in vitro (DIV)
Summary
Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. When bound to phospholipid membranes, a-synuclein adopts a helical conformation that mediates specific interactions with other proteins. Helical secondary structure when bound to phospholipid membranes [13]; this induced structure mediates specific interactions with other proteins [14]. An alternative b-sheet conformation of a-syn is associated with a number of neurodegenerative disorders, most prominently Parkinson’s disease (PD). Mutations in a-syn are associated with rare familial forms of Parkinson’s disease. These mutations include point mutations [17,18,19] and duplications or triplications of the gene SNCA [20,21,22], which encodes asyn protein
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