α-Synuclein Negatively Regulates Protein Kinase Cδ Expression to Suppress Apoptosis in Dopaminergic Neurons by Reducing p300 Histone Acetyltransferase Activity

Deregulation of PKCδ by α-synuclein protects against MPP⁺-induced cell death in dopaminergic N27 cells. A, Effects of downregulation of PKCδ by αsyn on MPP⁺-induced cell death in dopaminergic N27 cells. αSyn-expressing (Syn) and vector control (Vec) N27 cells were infected with lentiviruses expressing LacZ-V5 or PKCδ-V5 for 24 h. The cells were then exposed to MPP⁺ (300 μm) for 48 h. Cells were collected and assayed for DNA fragmentation (left panel) and caspase-3 activity (right panel). Data shown represent mean ± SEM from two independent experiments performed in quadruplicate (*p < 0.05; **p < 0.01; ***p < 0.001). B, MPP⁺-induced PKCδ proteolytic cleavage and its nuclear translocation were significantly diminished in αsyn-expressing N27 cells. αSyn-expressing (Syn) and vector control (Vec) N27 cells were exposed to MPP⁺ (300 μm) for 36 h. Cytoplasmic (C) and nuclear (N) fractions were prepared for immunoblotting analysis of PKCδ. LDH (cytoplasmic fraction) and Lamin B1 (nuclear fraction) were used as loading controls. C, Cytoplasmic localization of αsyn in αsyn-expressing N27 cells was not affected by MPP⁺ treatment. αSyn-expressing (Syn) and vector control (Vec) N27 cells were exposed to MPP⁺ (300 μm) for 36 h. Cells were either collected for preparation of cytoplasmic and nuclear extracts and immunoblotting analysis of αsyn (left panel) or stained and visualized under a Nikon TE2000 fluorescence microscope (right panel). Scale bar, 10 μm. A representative immunoblot and image of αsyn immunostaining (green) and Hoechst staining (blue) are shown.

Decreased PKCδ expression in nigral dopaminergic neurons in α-synuclein-overexpressing mice. A, Representative images of immunohistochemical analysis of PKCδ expression within nigral TH-positive neurons. Substantia nigra sections from nontransgenic control (non-tg) mice and αsyn-transgenic mice (htg) were stained with PKCδ polyclonal antibody (1:250 dilution) and TH monoclonal antibody (1:1800 dilution), followed by incubation with Alexa 568-conjugated (red; 1:1000) and Alexa 488-conjuated (green; 1:1000) secondary antibodies. Hoechst 33342 (10 μg/ml) was added to stain the nucleus. Confocal images were obtained using a Leica SP5 X confocal microscope system. Green, TH; red, PKCδ; blue, nucleus. The white arrows point to dopaminergic neurons with significant PKCδ staining. Scale bars: Left panel, 25 μm; right panel, 7.5 μm. Magnifications: Left panel, 63×; right panel, 430×. B, Quantification of the number of TH neurons containing colocalized PKCδ immunoreactivity was determined by blindly counting six fields and averaging. Values expressed as percentage of total TH neurons were mean ± SEM and representative for results obtained with three pairs of 6- to 8-week-old mice (***p < 0.001). C, To analyze the levels of αsyn in substantial nigra homogenates from transgenic mice overexpressing human wild-type αsyn and nontransgenic mice, substantial nigra homogenates were prepared from transgenic mice (htg) and nontransgenic mice (non-tg) and subjected to immunoblotting analysis of αsyn and β-actin. Representative immunoblot (left panel) and quantitation (right panel) of αsyn expression were shown. Approximately sixfold increase in αsyn expression in substantial nigra was found in transgenic mice. Data were shown as mean ± SEM; n = 6 (***p < 0.001).

α-Synuclein suppresses PKCδ transcription without affecting PKCδ protein or mRNA stability in N27 dopaminergic cells. A, Left, Pulse-chase analysis of stability of PKCδ protein. αSyn-expressing and vector control N27 cells were labeled with [³⁵S]methionine, and PKCδ protein was analyzed over 48 h as described in Materials and Methods. Right, The bands were quantified and expressed as percentage of amount present at time 0 h. The data plotted were fit to a one-phase exponential decay model using the nonlinear regression analysis program of Prism 4.0 software as follows: Y = span e^−Kt + plateau, where Y starts at span + plateau and decays with a rate constant K. The half-life of the protein was determined by 0.693/K. The square of the correlation coefficient (R²) is used as a measure of goodness-of-fit in regression analysis. Values are mean ± SEM of two independent experiments. B, The stability of PKCδ mRNA was not decreased in αsyn-expressing N27 cells. After treatment with ActD, total RNA was extracted for qRT-PCR analysis at selected time intervals. The relative abundance of PKCδ mRNA was expressed as a percentage of that present at time 0 h, and data plotted were fit to the one-phase exponential decay model. Values are mean ± SEM of three independent experiments performed in triplicate. C, The PKCδ promoter activation was attenuated in αsyn-expressing cells in reporter assays. Reporter pGL3-PKCδ carrying the PKCδ promoter or pGL3-Basic empty vector was transiently transfected into αsyn-expressing and vector control cells. Cells were collected 24 h after transfection and assayed for luciferase activity and β-galactosidase activity. Data were normalized and expressed as fold induction over the pGL3-Basic vector. Values are shown as mean ± SEM of three independent experiments performed in triplicate (***p < 0.001). D, The relative transcription efficiency of PKCδ was examined by quantitative nuclear run-on assay. Representative amplification plots for PKCδ mRNA (left panel) and β-actin mRNA (right panel) are shown. The change in fluorescence intensity (ΔRn) was plotted on the y-axis. The arrow shows the threshold (dashed lines). E, Quantitation of transcription efficiency. Data are expressed as fold change in the level of nascent run-on PKCδ mRNA in vector control cells and are shown as mean ± SEM of three independent experiments performed in triplicate (***p < 0.001).

Increased α-Synuclein expression suppresses PKCδ in part by blocking NFκB activation. A, Representative EMSA gel images show the direct binding of NFκB to the putative PKCδ NFκB sites. Competitive EMSA was conducted using labeled probe corresponding to the PKCδ NFκB site 1 (left panel) or the PKCδ NFκB site 2 (right panel) and indicated unlabeled oligos. B, Binding p50 and p65 to the NFκB sites on the PKCδ promoter. The nuclear extracts from vector control cells were incubated with excess of unlabeled self-oligos or indicated antibodies before adding the labeled probe (PKCδ NFκB site 1). A representative EMSA supershift gel from three independent experiments is shown. C, A representative EMSA gel image indicates the reduced binding of NFκB in vitro to the PKCδ NFκB site 1 in αsyn-expressing N27 cells. D, ChIP analysis of the in vivo binding of NFκB-p65 and p50 on the PKCδ promoter. After reversal of cross-linking, immunoprecipitated genomic DNA fragments were analyzed by PCR using primers designed to amplify the −103 to +60 region of PKCδ promoter. E, Knockdown of αsyn protein increased NFκB activity. αSyn-expressing cells were transient transfected with siRNA-αsyn and scrambled siRNA. Seventy-two hours after transfection, the cells were collected and subjected to EMSA analysis using the labeled probe corresponding to the PKCδ NFκB site 1. Mock transfection was also included as a negative control. F, Transfection of NFκB-p65 siRNA downregulated PKCδ expression in N27 cells. N27 cells were transfected with p65-siRNA and scrambled siRNA for 96 h, and cells were collected for Western blot analysis. Representative immunoblot (left panel) and quantitation (right panel) of p65 and PKCδ on whole-cell lysates in transfected cells. Data are shown as mean ± SEM of two independent experiments (*p < 0.05; **p < 0.01).

Effect of NFκB inhibition on the PKCδ immunoreactivity in the primary dopaminergic neurons. A, Primary midbrain cultures were treated with or without 100 μg/ml SN-50 for 24 h. Cultures were immunostained for TH (green) and PKCδ (red). The nuclei were counterstained by Hoechst 33342 (blue). Images were obtained using a Nikon TE2000 fluorescence microscope. Magnification, 60×. Scale bar, 10 μm. Representative immunofluorescence images are shown. The inset shows a higher magnification of the cell body area. B, Immunofluorescence quantification of PKCδ in TH-positive neurons. Fluorescence immunoreactivity of PKCδ was measured from TH-neurons in each group using MetaMorph software. Values expressed as percentage of control group are mean ± SEM and representative for results obtained from three separate experiments in triplicate (**p < 0.01).

α-Synuclein-induced blockade of NFκB activation is associated with decreased acetylation of p65 but does not correlate with nuclear translocation or protein levels of NFκB/IκBα. A, B, Nuclear translocation and abundance of NFκB/IκBα were not altered by overexpression of αsyn. Representative immunoblot of p65, p50, and IκBα levels on cytoplasmic and nuclear extracts (A) or whole-cell lysates (B) from αsyn (Syn) and vector control (Vec) cells. C, The p65 acetylation levels were reduced in αsyn cells. Whole-cell lysates was immunoprecipitated (IP) with p65 antibody. The resulting immunoprecipitates were blotted with anti-acetyl-lysine and anti-p65 antibodies. Densitometric quantitation of the ratio of band intensity of acetylated p65 and total p65 from two independent experiments (means ± SEM; **p < 0.01) is shown on the right. D, Sodium butyrate (NaBu) specifically enhanced PKCδ isoform expression in αsyn-expressing N27 cells. αSyn-expressing cells were treated with 1 mm NaBu and 50 μm caspase-3 inhibitor Z-DEVD-FMK, and cell lysates were prepared for blotting with specific anti-PKC isoforms (left panel) and anti-acetyl-lysine (right panel) antibodies.

α-Synuclein downregulates p300 proteins, resulting in decreased p300 HAT activity and inhibition of p300-dependent transactivation of PKCδ gene expression. A, Decreased p300 protein levels in αsyn-expressing cells. Representative immunoblot (left panel) and quantitation (right panel) of p300 and CBP on cytoplasmic and nuclear extracts from αsyn-expressing (Syn) and vector control (Vec) cells. Data are shown as mean ± SEM of two independent experiments (**p < 0.01). LDH (cytoplasmic fraction) or histone H3 (nuclear fraction) was used as loading control. B, Decreased p300 HAT activity in αsyn-expressing cells. Data were subtracted from background values that were measured in samples containing normal IgG, and then expressed as the percentage of HAT activity present in vector control cells. Values are shown as mean ± SEM of three independent experiments performed in triplicate (***p < 0.001). C, The in vivo binding of p300 on the PKCδ promoter was interrupted by overexpression of αsyn. After reversal of cross-linking, p300-immunoprecipitated genomic DNA fragments were analyzed by PCR using primers designed to amplify the −103 to +60 region of PKCδ promoter. D, Knockdown of p300 by siRNA-p300 decreased PKCδ levels in N27 cells. N27 cells were transfected with p300-siRNA and scrambled siRNA for 96 h, and cells were collected for Western blot analysis. Representative immunoblot (left panel) and quantitation (right panel) of p300 and PKCδ on nuclear extracts or whole-cell lysates in transfected cells. Data are shown as mean ± SEM of two independent experiments (*p < 0.05; ***p < 0.001).

Effect of p300 inhibition or activation on the PKCδ immunoreactivity in the primary dopaminergic neurons. A, Primary midbrain cultures at 7 d in vitro were treated with or without either 5 μm garcinol or 10 μm CTPB for 24 h. Cultures were immunostained for TH (green) and PKCδ (red). The nuclei were counterstained by Hoechst 33342 (blue). Images were obtained using a Nikon TE2000 fluorescence microscope. Magnification, 60×. Scale bar, 10 μm. Representative immunofluorescence images are shown. The inset shows a higher magnification of the cell body area. B, Immunofluorescence quantification of PKCδ in TH-positive neurons. Fluorescence immunoreactivity of PKCδ was measured from TH-neurons in each group using MetaMorph software. Values expressed as percentage of control group are mean ± SEM and representative for results obtained from three separate experiments in triplicate (*p < 0.05; **p < 0.01).

Decreased p300 level within neurons of the substantia nigra in αsyn-overexpressing mice. Representative images of immunohistochemical analysis of p300 expression within nigral TH-positive neurons. Substantia nigra sections from nontransgenic control (non-tg) mice and αsyn-transgenic mice (htg) were stained with p300 polyclonal antibody (1:350 dilution) and TH monoclonal antibody (1:1800 dilution), followed by incubation with Alexa 568-conjugated (red; 1:1000) and Alexa 488-conjugated (green; 1:1000) secondary antibodies. Hoechst 33342 (10 μg/ml) was added to stain the nucleus. Confocal images were obtained using a Leica SP5 X confocal microscope system. The white arrows point to dopaminergic neurons with significant nuclear p300 staining. Green, TH; red, p300; blue, nucleus. Scale bars: Left panel, 25 μm; right panel, 7.5 μm. Magnifications: Left panel, 63×; right panel, 250×.