Alpha-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells

Abstract

Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated alpha-synuclein in oligodendrocytes. p25alpha is an oligodendroglial protein that potently stimulates alpha-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25alpha and alpha-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in alpha-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of alpha-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic alpha-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.

Figures

FIGURE 1.

Coexpression of α-synuclein and p25α causes retraction of microtubule to the perinuclear region. A and B, α-syn-expressing OLN-AS cells were transiently transfected with p25α for 12 h (A) or 24 h (B) and subjected to immunofluorescence microscopy using anti-α-tubulin and anti-p25α antibodies or to phase contrast microscopy (Bj). Overlay with nuclear DAPI staining is shown in c, f, and i. The gradual MT retraction from processes to the perinuclear region upon coexpression of p25α and α-syn is evident. Scale bars, 20 μm (apply to panels A and B). MT retraction to the perinuclear region is quantified in panel C and used as a measure in the following figures. C, development of MT retraction among p25α-transfected OLN-AS cells (filled circles) and OLN-t40 cells (open circles) and mock-transfected OLN-AS cells (filled triangle) was quantified by calculating the percentage of p25α expressing cells, which demonstrated MT retraction. The points represent the mean ± 1 S.D. from five microscopic fields in one of three representative experiments. Note the absent MT retraction in mock-transfected OLN-AS cells and in p25α-transfected OLN-t40 cells. D, the expression of p25α in OLN-AS cells was analyzed by Western blotting using anti-p25α. α-tubulin was included as a loading control. E, quantification of the ratio between p25α and tubulin from panel D.

FIGURE 2.

Retraction of cellular processes is selective for coexpression of α-synuclein and p25α. A and B, OLN-93 (A) and tau-expressing OLN-t40 cells (B) were transiently transfected with human α-syn and empty vector (a-c), p25α and empty vector (d-f), and α-syn and p25α (g-i). Cells were examined 24 h after transfection by immunofluorescence microscopy using anti-α-syn and anti-p25α antibodies. Single transfections with either protein resulted in a diffuse distribution in cellular processes and cytoplasm (A and B, a-f). Coexpression of the two proteins resulted in retraction of the proteins from the cellular processes (A and B, g-i). C, OLN-AS cells retract their processes upon expression of p25α (d-f), whereas transfection with an empty vector (a-c) has no effect. Immunofluorescence microscopy was performed using rabbit anti-α-syn (ASY1), rat anti-p25α antibodies, and DAPI staining of the nuclei. D, expression of human β-synuclein in the OLN-AS cells for 24 h did not cause MT retraction. The β-synuclein-expressing cells were analyzed using anti-α-tubulin and anti-β-synuclein as primary antibodies. Note the retraction of cellular processes is selective for coexpression of α-syn and p25α (A, g-i; B, g-i; C, d-f) and does not occur upon coexpression of α-syn and tau40 (B, d-f) and α-syn and β-synuclein (D, a-c). Scale bars, 20 μm (apply to panels A-D). E, cell lysates were prepared 24 h after transfection, and 20 μg of protein was analyzed by Western blotting using ASY1 and anti-p25α antibodies. α-Tubulin was included as a loading control.

FIGURE 3.

A low concentration of α-synuclein is sufficient for p25α-induced microtubule retraction. A and B, OLN-93 cells were cotransfected with 1 μg of p25α vector and the indicated concentrations of α-syn vector complemented with empty vector control to assure equal DNA load (in 6-well plates). A, cell lysates were prepared 24 h after transfection, and 30 μg of protein was analyzed by Western blotting using ASY1 and anti-p25α antibodies. α-Tubulin was included as a loading control. B, quantification of the ratio between α-syn and tubulin from panel A (open circles) and quantification of cotransfected cells showing MT retraction (filled circles) are shown as the mean ± 1 S.D. from five microscopic fields in one of three representative experiments. C and D, OLN-AS cells were transfected with siRNA targeting human α-syn or with a non-targeting control (siControl). The expression level of α-syn was analyzed by immunofluorescence microscopy (C) and Western blot analysis (D) 96 h after siRNA-transfection. One representative experiment of three is demonstrated. E, OLN-AS cells were transfected with siRNA for 96 h followed by transfection with p25α. The number of cells displaying MT retraction was quantified 24 h after transfection with p25α. Bars represent the mean ± 1 S.D. of three independent experiments. RNAi-mediated silencing of α-syn causes a significant reduction in the amount of p25α-positive cells displaying MT retraction (p < 0.05 with respect to untreated cells).

FIGURE 4.

Microtubule retraction is dependent on phosphorylation at Ser-129 in α-synuclein. A, OLN-93 cells were transfected with α-syn wt, α-syn S129A, or α-syn S129D for 24 h, and 20 μg of protein was subjected to immunoblotting using ASY1 and anti-α-tubulin antibodies. B, OLN-93 cells were transfected with p25α or α-syn (wt, S129A, or S129D) or cotransfected with p25α and the α-syn variants as indicated. At 24 h post-transfection cells were subjected to immunofluorescence microscopy with ASY1, anti-p25α, and anti-α-tubulin antibodies. MT retraction in the single and cotransfected cells was quantified, and bars represent the mean ± 1 S.D. of three independent experiments. Coexpression of α-syn wt and p25α or α-syn S129D and p25α caused significant MT retraction (p < 0.05 compared with cells cotransfected with p25α and empty vector), whereas coexpression of α-syn S129A and p25α had no significant effect on MT retraction. C, the kinase inhibitor DMAT does not affect α-syn expression as revealed by ASY1 and anti-β-actin immunoblotting of lysates from OLN-AS cells. D, OLN-AS cells transfected with p25α were treated with the indicated concentrations of DMAT and subjected to anti-p25α and anti-α-tubulin immunofluorescence microscopy to quantify MT retraction. Bars represent the mean ± 1 S.D. of three independent experiments. DMAT caused a dose-dependent attenuation of the MT retraction (p < 0.05 with respect to untreated cells). E and F, anti-Ser(P)-129 (E) and ASY1 (F) immunoblotting (WB) of α-syn immunoprecipitates from OLN-AS cells transfected with an empty vector or with p25α in the presence or absence of 5 μm DMAT. IgG bands are indicated by as asterisk. Note the enhanced anti-Ser(P)-129 (E)- and ASY1 (F)-positive high molecular weight smear in cells coexpressing α-syn and p25α that is reduced by treatment with DMAT. Molecular size markers are shown to the left. The experiment was repeated twice with similar results.

FIGURE 5.

Inhibition of α-synuclein aggregation attenuates microtubule retraction. A, OLN-AS cells were pretreated with cell impermeable (ASI1) and cell-permeable (ASI1D and scrambled ASI1D(s)) peptide inhibitors of α-syn aggregation for 1 h before transfection with p25α. Alternatively, cells were treated with Congo Red for 7 days or baicalein for 1 h before transfection with p25α. MT retraction was quantified 24 h after transfection. Bars represent the mean ± 1 S.D. of three independent experiments. Congo Red, baicalein, and the cell-permeable inhibitor ASI1D caused a significant reduction in the number of p25α-positive cells displaying MT retraction (p < 0.05 with respect to untreated cells). B, internalization of biotinylated ASI1D was confirmed by ApoTome sectioning at the midnuclear plan. Cells were treated with biotinylated ASI1D (upper figure) or ASI1 (lower figure) for 1 h at 37 °C, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were incubated with Alexa Fluor 488-conjugated streptavidin and visualized under a fluorescence microscope equipped with an ApoTome for optical sectioning. Scale bar, 20 μm.

FIGURE 6.

Microtubule retraction precedes development of apoptotic characteristics. A, OLN-AS cells were treated with a peptide aldehyde caspase-3 inhibitor Ac-DEVD-CHO (20 μm) before transfection with p25α. The number of transfected cells with MT retraction was quantified 24 h after transfection. Bars represent the mean ± 1.S.D. of three independent experiments. Inhibition of caspase-3 caused a significant reduction in MT retraction (p < 0.05 with respect to untreated cells). B, lysates from OLN-AS and OLN-t40 cells transfected with either mock or p25α vector for 24 h were analyzed for caspase-3 activity using Ac-DEVD-amido-4-methylcoumarin as substrate. Concentrations of released fluorescent products were measured in arbitrary units, and bars represent the mean ± 1 S.D. of triplicate. One of three representative experiments is shown. The presence or absence of the caspase-3 inhibitor, Ac-DEVD-CHO (20 μm), is indicated by a + or - below the bar chart. Cells treated with camptothecin (CAMP) were included as a positive control. OLN-AS cells expressing p25α demonstrate a significant increase in caspase-3 activity (p < 0.05) as compared with mock-transfected OLN-AS cells and p25α-transfected OLN-t40 cells. C, OLN-AS cells were transfected with p25α for 24 h and stained with anti-p25α and an antibody specific for active caspase-3. D, OLN-AS cells were transfected with either an empty vector (left) or p25α vector (middle and right) and stained with Annexin-V-fluorescein isothiocyanate and DAPI 24 h after transfection. E, OLN-AS cells were transfected with either an empty vector (left) or with p25α (middle and right). Apoptotic cells were identified by a condensed (middle) or fragmented (right) nuclei. Scale bars, 20 μm. F and G, the population of OLN-AS cells with apoptotic nuclei (F) and MT retraction (G) was quantified 24, 48, and 72 h after p25α-transfection in the absence or presence of ASI1D, DMAT, and DEVD. Points represent the mean ± 1 S.D. from five microscopic fields in one of three representative experiments. The development of MT retraction during the first 24 h is displayed in Fig. 2.

FIGURE 7.

Model for development of α-synuclein-mediated cytotoxicity. Proaggregatory factors such as p25α nucleate monomeric α-syn and thereby convert the monomers into better substrates for Ser-129-reactive kinases. The Ser-129 phosphorylation increases the nucleating activity of the α-syn aggregates, which favors the formation of cytotoxic Ser(P)-129 oligomers. These oligomers subsequently affect vital cellular signaling pathways that ultimately initiate slow activation of caspase-3 and its downstream prodegenerative effects comprising early MT dysfunction and subsequent apoptosis.