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

FAS is upregulated in human MSA brain.

<p><b>A, </b><b>B,</b> Immunohistochemical staining for FAS in normal controls. In normal controls there were only occasional FAS-positive cells shown here in (A) the inferior temporal cortical white matter and (B) grey matter. Insert in (A) shows a FAS-positive cell resembling a microglia. <b>C–H,</b> Immunohistochemical staining for FAS in MSA cases. MSA tissue contained numerous FAS-positive glial cells shown here in the inferior temporal cortical (C) white matter and (D) grey matter. Arrows indicate FAS-positive oligodendrocytes, and arrowheads indicate FAS-positive astrocytes or microglia. Inserts in (C) and (D) shows enlarged view of boxed regions showing FAS-positive oligodendrocytes (judged by their nuclear morphology). The oligodendrocyte in (D) contains an inclusion. E, FAS-positive glial cells in the cerebellar white matter. Boxed region in (E) is enlarged in (F) showing oligodendrocytes. G, FAS-positive cells in the pons. Arrow indicates a FAS-positive glial cell resembling a GCI-bearing oligodendrocyte. Arrowheads indicate FAS-positive astrocytes and microglia. Boxed region in (G) is enlarged in (H). <b>I,</b> Brain tissue (precentral gyrus white matter) from human MSA (<i>n</i> = 8) and control cases (<i>n</i> = 10) was sequentially extracted for TBS and SDS soluble proteins and the SDS soluble fraction were used for further analysis. Four MSA and four control cases are shown. Proteins (20 μg) were resolved by SDS-PAGE and analyzed by immunoblotting using antibodies against α-syn, p25α and FAS. Actin was included as a loading control. The molecular sizes (kDa) of the presented bands are indicated to the left. There was a significant increase in SDS-soluble FAS protein in MSA cases compared to controls (<i>p</i> = 0.043).</p

α-synuclein dependent degeneration in OLN-93 cells requires FAS signaling and caspase-8 activation.

<p>OLN-t40-AS cells stably expressing human α-syn were treated with peptide aldehyde inhibitors (20 µM) against caspase-3 (Ac-DEVD-CHO), caspase-8 (Ac-IETD-CHO), caspase-9 (Ac-LEHD-CHO) and FAS-blocking antibody (ZB4) (1 µg/ml) 1 h prior to transfection with p25α. MT retraction was quantified my immunofluorescence microscopy 24 h after transfection. Bars represent the mean ± standard error of mean (SEM) from three independent experiments. Inhibition of caspase-3, caspase-8 and FAS but not caspase-9 caused a significant reduction in MT retraction as compared with the control cells (<i>p</i><0.05 with respect to untreated cells).</p

α-synuclein expressing oligodendrocytes are sensitized to FAS-dependent toxicity.

<p><b>A, </b><i>In vitro</i> differentiation of (PLP)-α-syn tg oligodendrocytes. Oligodendrocyte progenitor cells were isolated from tg mouse forebrains and cultured for the indicated times. Cells were fixed for double-label immunostaining with antibodies against A2B5, NG2, O4, GalC and MBP (green; left panels) as well as monoclonal 15G7 against human α-syn (red; right panels). Differentiated cells (7 DIV) were immunostained with an antibody against p25α and visualized by phase contrast microscopy. Scale bars, 10 µm. <b>B,</b> Human α-syn and eGFP tg oligodendrocytes were treated for 24 h with DMSO (control), sFASL, mFASL or preincubated for 30 min with FAS-blocking antibody before challenge with mFASL. Cells were fixed and labeled with O4 and Hoechst. mFASL treatment induced oligodendrocyte cell death as determined by apoptotic nuclei with α-syn oligodendrocytes being significantly more sensitive to FAS mediated cell death than eGFP oligodendrocytes. Data represent mean ± SEM of total tg oligodendrocytes from three independent experiments. Student <i>t</i> test (<i>n</i> = 3): *<i>p</i><0.0001 compared with untreated cultures; #<i>p</i><0.001 compared with eGFP tg cultures exposed to the same challenge. Western blots prepared from lysates of wild-type and α-syn tg oligodendrocyte cultures were sequentially probed with monoclonal anti-FAS and anti-α-tubulin antibodies (insert).</p

FAS colocalizes with p25α and α-synuclein in human MSA brain.

<p>Double labeling immunofluorescence using FAS (red) and p25α (green) antibodies (A–F) and FAS (red) and α-syn (green) antibodies (G–L) in putamen of an MSA case. Protein colocalization is shown as yellow in merged images (C, F, I, L). A–C, Identification of p25α-positive/FAS-negative GCIs (arrows) in oligodendroglia. D–F, Colocalization of FAS and p25α within GCI-like structures but not in small punctate cytoplasmic granules. G–L, FAS and α-syn colocalized in 4% of the GCIs (J–L) but the majority of α-syn-positive GCIs did not colocalize FAS (G–I, arrows indicate α-syn-positive/FAS-negative inclusions).</p