Dynamic transport and localization of alpha-synuclein in primary hippocampal neurons

Abstract

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.

Figure 1

ASGFP localizes to the presynaptic terminal by 17DIV. Primary rat E18 hippocampal neurons were transfected with either ASGFP (top and middle rows, green) or eGFP (bottom row, green). After 17 days in vitro (DIV), cells were fixed and immunostained for MAP2 (top row, red), or synapsin I (middle and bottom rows, red), and visualized for immunofluorescence. Scale bar = 10 μm.

Figure 2

Schematic representation of α-synuclein/eGFP constructs. (A) Schematic of wild-type α-syn with C-terminal eGFP fusion; coding exons are depicted as blue boxes numbered 2 to 6. Positions of two PD-associated mutations (A30 and A53) are indicated, and black lines represent the predicted secondary structure in the presence of lipid. (B) Exon deletion mutants; crosshatched boxes represent deleted regions.

Figure 3

Localization pattern of exon-deletion mutants. Primary rat E18 hippocampal neurons were transfected with α-syn variant constructs. After 17 DIV, cells were fixed and immunostained for synapsin I. The left column shows the eGFP signal from the various α-syn constructs (green). The middle employs the "find edges" filter in Adobe Photoshop, which represents color transitions as lines. The right column shows a merge of the eGFP signal from the various α-syn constructs (green) with the synapsin I staining (red).

Figure 4

Quantification of localization. Bar graph showing the localization index of each construct expressed in neurons. The localization index is a ratio of average signal intensity from boutons versus average signal intensity from axonal processes. The mean localization index of each mutant (n = 15 cells) was compared to ASGFP using pairwise t-test with Bonferroni correction for repeated measures (*p < 0.0001, **p = 0.0002).

Figure 5

ASGFP transport behavior. (A) Time lapse fluorescent images of ASGFP transport particles in rat primary hippocampal neurons visualized at 10 DIV. The single-headed arrow indicates an ASGFP structure that moves along the axon, while the double-headed arrow indicates an ASGFP structure moving in the opposite direction. (B) Kymograph of the full time lapse series, with the time window depicted in A noted on top (46-81s). The particles are labeled as in panel A. One of the particles (double-headed arrow) is seen to move continuously in one direction, while other particle (single-headed arrow) moves in the opposite direction, pauses, and then bifurcates into stationary (s) and motile (m) particles.

Figure 6

Examples of ASGFP transport behavior. Kymographs illustrating examples of ASGFP transport behavior. (A) One particle is clearly seen to bifurcate into two distinct particles (bold arrow). (B) Three starting particles briefly fuse to become one particle (bold arrow) before bifurcating again into two particles. (C) One particle (bold arrow) moves bidirectionally in and out of a stationary accumulation of ASGFP (fine arrow) (D) One particle moves without stopping or changing direction (bold arrow) (E) One particle gradually emerges from the background (bold arrow). (vertical scale bar = 50 μm, horizontal scale bar = 30s).

Figure 7

Velocity distribution for ASGFP and mutants. Velocities during periods of movement were calculated from kymographs, and mean ± SEM plotted as a percentage of ASGFP velocity. A53TGFP velocity was decreased relative to ASGFP (*p = 0.0033, pairwise t-test with Bonferroni correction; n>50 movies per group).

Figure 8

Quantitation of motile particles. Primary hippocampal neurons were transfected at 7 DIV. Kymographs were obtained from the proximal axon segment at 10-14 DIV, and particles displaying >10 μm continuous movement were counted and normalized to the length of the axon segment. (*p < 0.02 for the comparison with ASGFP, one-way ANOVA with post-hoc Tukey test, n ≥ 9 cells per group)