Effect of spermidine on the appearance of force-distance curves with A30P alpha-synuclein.

<p>Superposition of representative force-distance curves measured upon probing of interactions between A30P alpha-synuclein molecules: A) in the absence of spermidine (40 curves), B) with addition of 5 mM spermidine (58 curves).</p

Interaction model of alpha-synuclein molecules.

<p>The model describes three major peaks in the contour length histograms as identical sites in each monomer interacting with each other with the formation of a dimer. A) Position of the interacting site further from the C-terminus (point of attachment) results in longer contour length value. B) Positions at the beginning of the detected interaction sites. Colored arrows correspond to three detected interaction sites schematically shown in A), and the black arrow shows the position of the A30P mutation in alpha-synuclein; C), D) and E) superposition of representative force-distance curves for the detected rupture events corresponding to L_C1, L_C2 and L_C3, red, green and blue lines on the graphs are WLC curves calculated with L_C values from Fig. 4.</p

Effect of spermidine on the appearance of force-distance curves with wild-type alpha-synuclein.

<p>Superposition of representative force-distance curves measured upon probing of interactions between wild type alpha-synuclein molecules: A) in the absence of spermidine (22 curves), B) with addition of 5 mM spermidine (26 curves).</p

Schematic representation of the experimental approach.

<p>A) The unstructured form of alpha-synuclein immobilized on the tip and a substrate reveals no interaction. B) Misfolding of alpha-synuclein results in the formation of aggregation-prone conformation with elevated propensity of intermolecular interactions. C) A representative force-distance curve measured in the absence of spermidine. D) A representative force –distance curve with a rupture event in the presence of spermidine: (1) an adhesion peak due to short-range non-specific interactions between the tip and the surface, (2) gradual increase in force characteristic of polymer stretching, (3) complete rupture at 110 pN and (4) region where tip comes free from the surface. The inset shows worm-like chain fitting yielding contour length of 31 nm.</p

α‑Synuclein Misfolding Assessed with Single Molecule AFM Force Spectroscopy: Effect of Pathogenic Mutations

Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson’s disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early onset PD; however, the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central nonamyloid-beta component (NAC) of the protein but rather expand beyond this segment. All three mutations alter the protein’s folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed

Complex force-distance curves with multiple rupture peaks detected with WT alpha-synuclein (A, B) and A30P mutant (C, D).

<p>Red, green and blue lines on the graphs are calculated WLC curves using maxima of Gaussians determined from contour length distributions corresponding to 26, 36 and 47 nm (WT) and 22, 34 and 49 nm (A30P). E) Pie charts showing relative contribution of multiple rupture events to total rupture events: 31% (WT) and 15% (A30P).</p

[Around the World, Or the Belgian Conquest]

Figure S1. ENSA does not have a pronounced effect on the binding of aSyn to phospholipid membranes. Figure S2. A29E aSyn fails to elicit membrane disruption. Figure S3. ENSA exhibits no membrane disruption activity on its own. Figure S4. Results of Western blot analysis showing equal expression levels of WT ENSA and S109E in primary midbrain cultures. Figure S5. WT ENSA and S109E do not elicit neurotoxicity when expressed alone or in combination with β-gal. Figure S6. Western blot image showing a trend towards reduced ENSA expression levels in the substantia nigra region of PD patients versus non-diseased individuals. Table S1. P values for vesicle permeabilization data in Fig. 3a and b. Table S2. P values for vesicle permeabilization data in Fig. 3c and d. Table S3. Summary of demographic information for donors of substantia nigra samples. (DOCX 421 kb

ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.

<p><b>A.</b> ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 µg/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087133#pone.0087133.s001" target="_blank">file S1</a>). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 μ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (n = 3+/− s.e.m *p≤0.001 versus no compound Tet-induced sample). <b>B.</b> Microglia isolated from postnatal day 1 to 3 pups from hSNCA^E46K transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 µM ELN484217 or ELN484228 followed by addition of 10 µm beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3+/− s.e.m *p≤0.001). <b>C.</b> ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (‘control’) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 µM ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. n = 3 for neuron viability analysis; n = 160–206 for neurite length analysis. *p-value≤0.05, ***p-value≤0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. <b>D.</b> ELN484228 reduces translocation of αSyn to the phagocytic cup<b>.</b> To assess αSyn translocation, H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline for 24 hours; cells were then stimulated with 4 μ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. <b>E.</b> ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (∼21DIV) grown in serum-free conditions were treated for 24 hours with 1 µM ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 µm. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/− SEM, n = 1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2–3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.</p