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

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

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 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

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