Kragh CL , Fillon G , Gysbers A , Hansen HD , Neumann M , Richter-Landsberg C , Haass C , Zalc B , Lubetzki C , Gai WP , Halliday GM , Kahle PJ , Jensen PH .

R24AA012725 NIAAA NIH HHS , Medical Research Council

	Figure 2. α-synuclein expressing oligodendrocytes are sensitized to FAS-dependent toxicity.A, In vitro 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, 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 t test (n = 3): *p<0.0001 compared with untreated cultures; #p<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).
	Figure 3. FAS is upregulated in human MSA brain.A, 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. C–H, 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). I, Brain tissue (precentral gyrus white matter) from human MSA (n = 8) and control cases (n = 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 (p = 0.043).
	Figure 4. FAS colocalizes with p25α and α-synuclein in human MSA brain.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).
	Figure 1. α-synuclein dependent degeneration in OLN-93 cells requires FAS signaling and caspase-8 activation.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 (p<0.05 with respect to untreated cells).

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