Characterizing the Dynamics of α‑Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Soluble oligomers formed by α-synuclein (αSN) are suspected to play a central role in neuronal cell death during Parkinson’s disease. While studies have probed the surface structure of these oligomers, little is known about the backbone dynamics of αSN when they form soluble oligomers. Using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of αSN (residues 94–140) underwent isotopic exchange very rapidly, demonstrating a highly dynamic region in the oligomeric state. Three regions (residues 4–17, 39–54, and 70–89) were strongly protected against isotopic exchange in the oligomers, indicating the presence of a stable hydrogen-bonded or solvent-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18–38 and 55–70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35–95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance analyses performed on αSN fibrils and indicated a possible zipperlike maturation mechanism for αSN aggregates. We find the protected N-terminus (residues 4–17) to be of particular interest, as this region has previously been observed to be highly dynamic for both monomeric and fibrillar αSN. This region has mainly been described in relation to membrane binding of αSN, and structuring may be important in relation to disease

Validation of monomer and oligomer preference of αSN interacting proteins.

<p>Proteins pulled down by monomer αSN (M), oligomer αSN (O), and buffer control (B) from porcine (A-F) and human (G-I) brain extracts were analyzed by immunoblotting using antibodies against antigens selected among the monomer and oligomer binding proteins. Monomer binding antigens were myelin proteolipid protein (mPLP) and Abl interactor 1 (Abl1) and oligomer binding proteins were glial fibrillary acidic protein (GFAP), glutamate decarboxylase 2 (GAD2), and synapsin 1 (Syn1). VAMP-2 was tested because it has been reported to bind αSN, although it was not detected in our proteomic analysis. One representative of three experiments is presented for porcine αSN binding proteins (A, C, E), and the quantification of the three experiments is presented in panels B, D, F. The quantification of bands was performed after subtracting the non-specific signal in the buffer control from the specific bands in monomer and oligomer samples. Bars represent mean ratio between monomer and oligomer ± S.D. of the three replicates. The values for binding to monomer and oligomer were compared by Student’s t-test and the resulting p-values are listed above the bars. * Indicates that the band intensity from oligomer did not differ significantly from background making quantifications impracticable. In order to ensure that the interaction were not due to species differences between human and porcine proteins we conducted validations in human brain extracts. One representative of two experiments is presented for each validated protein. The validation for both porcine and human of mPLP, Abl1, Syn1 and VAMP-2 was conducted in the LP2 fraction enriched in synaptic vesicle and the validation to GFAP and GAD2 in the LS1 fraction of synaptosomal lysate.</p

Possible molecular pathways initiated by αSN in disease.

<p>Under normal conditions αSN is predominantly located in nerve terminals (blue). During disease αSN undergo aggregation and this lead to novel conformation-dependent interactions (red), which represents a gain of function. In addition, αSN species (monomeric and oligomeric) are concentrated at abnormal sites, like axons and the cell body, or in astrocytes and oligodendrocytes, which give rise to novel interactions because new partners are introduced (green). Finally, an abnormal sorting and aggregation leads to a loss of, or reduced normal αSN concentration in nerve terminals where critical monomer specific interactions will be compromised thus representing a loss of function.</p

Detection of oligomeric a-syn in synaptosomes isolated from whole brains of mThy-h-a-syn transgenic mice (ASO) and verification of folding dependent epitopes.

<p>Oligomer a-syn (A.) and total a-syn (B.) levels are measured in whole brain synaptosomes from adult ASO (n = 3) and WT (n = 3) mice that were extracted in detergent containing RIPA buffer and analyzed by ELISA for their a-syn content. <b>A</b>. The oligomer a-syn ELISA reveals increased oligomer levels in the ASO mice compared to the WT mice. The oligomer signal is aggregate specific because no increase is obtained by spiking the sample with increasing amounts of recombinant monomer standards <b>B</b>. The total level of a-syn is increased in ASO mice compared to WT and spiking the sample with the same recombinant monomeric a-syn standard as used in panel A increases the level of total a-syn when more than 5 ng/ml monomer is added. <b>B</b>. Bars in A and B represents standard deviation of three biological replicates (ASO and WT). Significance is marked with an asterisk: *p<0.05; **p<0.005 and indicate in (A.) and (B.) the given sample related to wt. Results were compared using one-way ANOVA followed by Turkey’s <i>post hoc</i> test for multiple comparison. <b>C.</b> Folding specificity of the MJF14 binding to a-syn species in synaptosomes was analyzed by dot blotting in the absence and presence of denaturing 50% formic acid (FA). Dilution of RIPA extracts of ASO mouse brain synaptosomes was applied to the dot blots in the absence and presence of treatment with denaturing 50% formic acid (FA). The filters were tested for binding of MJF14-6-4-2 to detect the aggregate dependent epitope (left panel-oligomer) and ASY-1 to detect total a-syn (right panel-total a-syn). Right dot blot shows the total level of a-syn, which is equal in the two samples treated or untreated with FA. FA treatment removes the epitope for the MJF14-6-4-2 antibody but not the ASY-1 epitopes confirming the folding specificity of the MJF14-6-4-2 epitope.</p

Subcellular localization of the αSN interacting proteins.

<p>A total of 178 proteins were identified as αSN interacting proteins and they were grouped in proteins preferentially binding monomer αSN (MP, N = 10, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t001" target="_blank">Table 1</a>), oligomers (OP, N = 76, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t002" target="_blank">Table 2</a>) and proteins not displaying any preferences (NPB, N = 92, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116473#pone.0116473.t003" target="_blank">Table 3</a>). They were grouped based on their subcellular localization as described by their principal localization in the Uniprot database to demonstrate the aggregation state of αSN have potential for significantly changing its cellular targets.</p

Preferential αSN monomer interacting proteins.

<p>Preferential αSN monomer interacting proteins.</p

Specificity and affinity of the MJF14-6-4-2 antibody.

<p><b>A.</b> Monomers and oligomers were isolated by gel filtration and eluted in two distinct fractions with peak values corresponding to 60 kDa and 800 kDa, respectively (grey broken line). The absorbance profile at 214 nm is indicated at the right ordinate axis. To validate the specificity of the oligomer ELISA we incubated antibody coated wells with the same concentration of monomer and oligomer (9 ng/mL) and used a monoclonal anti-a-syn antibody as second antibody. Bound antibody was detected by anti-mouse HRP and the TMB color change was measured as absorbance at 450 nm. The ELISA gives a strong signal for oligomers whereas there is only a negligible signal from the monomer sample (black bars). Background absorbance was subtracted. The ELISA signal (absorbance at 450 nm) is presented at left ordinate axis. <b>B.</b> We compared the aggregate specificity of the MJF14-6-4-2 antibody with the well characterized polyclonal antibody FILA-1 using dot blotting analysis. Dilutions of monomers (M) and oligomers (O) were immobilized and tested for their antibody binding. The non-conformation dependent monoclonal a-syn antibody (BD) (0.5 μg/ml) demonstrated equal loading of monomer and oligomer. MJF14-6-4-2 (Abcam) (2.2 ng/ml) and FILA-1 (3.5 μg/ml) antibodies display the same extend of selectivity for the aggregated a-syn. <b>C.</b> The affinity of MJF14-6-4-2 antibody was assessed by a direct ELISA on oligomer coated wells. The MJF14-6-4-2 (15.6 ng/ml) antibody was pre-incubated overnight with increasing concentrations of monomer or oligomer before being applied to the oligomer containing wells. The concentration of oligomer that gave 50% inhibition was measured to be 42 ng/ml. The K_D-value of MJF14-6-4-2 was calculated to be 2.9 · 10⁻¹⁰ M based on the assumption that 10 a-syn monomers are required to generate a folding specific MJF14-6-4-2 epitope. Both A and C show one representative experiment of 3. Standard deviations represent three technical replicates.</p

A-syn oligomer ELISA sensitivity and specificity.

<p><b>A.</b> Test of three different concentrations (0.125, 0.0625, and 0.03125 μg/ml) of capture antibody MJF14-6-4-2 (MJF) for monomeric and oligomeric a-syn for the oligomer ELISA. 0.0625 μg/ml is chosen as the optimal concentration for MJF14-6-4-2 based on sensitivity and specificity from monomeric a-syn. <b>B.</b> The oligomer ELISA detects oligomeric a-syn in a linear range from 0.25–20 ng/ml oligomer a-syn with the use of MJF14-6-4-2 as capture antibody and the anti-a-syn (BD) as detection antibody (R² = 0.99). Monomeric a-syn is not detected by the oligomer ELISA in this range. Negative control (PBS) shown in light grey. <b>C.</b> The graph from C. zoomed in on the lower part of the X-axis. The oligomer ELISA detects down to 0.25 ng/ml oligomeric a-syn (P = 0.001). <b>D.</b> Determination of inter-assay coefficient of variation (CV). Comparison of results from four different runs is used to determine CV for the oligomer ELISA. CV is calculated for 6 different concentrations of oligomers (1.25 ng/ml– 40 ng/ml) resulting in 6 different CVs values all below 6%—in average 4,5%. <b>E.</b> Recovery by recombinant a-syn oligomers spiked into WT mouse brain synaptosomes. Mouse brain synaptosomes are spiked with different amounts of oligomers (1, 2, 3, and 5 ng/ml). Expected increase in absorbance is calculated from an internal series of controls, and the recovery rate is calculated for each concentration of spiked amount of oligomer. All recovery rates are above 95%—in average 98%. Data are an average of 4 different experiments run in triplicates. Data are compared to unspiked for significance. <b>F.</b> Validation that a-syn oligomer signal is caused by conformational epitopes. Treatment with 8M urea inhibits the a-syn oligomer signal on the oligomer ELISA. Higher concentrated oligomers are treated for 3 hours with 8M urea, and then diluted to 3mM urea before addition to the ELISA. <b>G.</b> To validate that the urea treatment does not affect the binding capability of the ELISA or/and the antibodies used, the experiment was run with longer incubation time, an oligomer concentration of 2.5 ng/ml, and low amounts of urea equivalent to the amount of UREA present in the urea treated oligomers after dilution (urea concentration during running conditions = 3 mM). 8M urea treatment reduces the oligomer signal, whereas 3mM urea treatment does not remove the oligomer signal. “None” represents non-treated a-syn oligomers, whereas background signal from PBS without a-syn is shown by the dotted grey line. Both G. and H. show one representative experiment of 3. Standard deviations represent three technical replicates. Significance for all parts of figure is marked with an asterisk: *p<0.05; **p<0.005. Results were compared using one-way ANOVA followed by Turkey’s <i>post hoc</i> test for multiple comparison.</p

Time dependent development of a-syn oligomers in a non-mitotic a-syn transgenic cell model.

<p>A-syn SH-SY5Y Tet-off cells were differentiated with retinoic acid to induce a non-mitotic state where after a-syn expression was induced by removal of doxycycline. PBS background is shown by the dotted grey line. <b>A.</b> Measurement of a-syn by the total a-syn ELISA in RIPA extracts demonstrated a linear increase in cellular a-syn that demonstrates approximately a doubling of a-syn from day 5 to day 10. <b>B.</b> Analysis of the RIPA extracts by the oligomer ELISA demonstrates no detectable oligomers after 5 days but a significant increase after 10 days. Bars represents standard deviation of 3 biological experiments (all run in three technical replicates). The Student’s unpaired t-test was used to compare 5 and 10 days. Significance is marked with an asterisk: *p<0.05; **p<0.005. For A., 5 days is also significantly different from PBS background, whereas 5 days in B. cannot be distinguished by PBS.</p