Journal: The Journal of Neuroscience

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

doi: 10.1523/JNEUROSCI.2507-13.2014

Figure Lengend Snippet: Apposition of ER and mitochondria in HeLa cells. A, Examples of colocalization of ER labeled with GFP-Sec61-β (green), and mitochondria labeled with pDsRed2-mito (red), in HeLa cells transiently expressing WT- and A30P-α-syn. B, Quantitation of colocalization (as in A) by ImageJ analysis, normalized to the baseline level of apposition value in EV cells (baseline = 100; average of 4 experiments ±SD). Asterisk denotes significance versus EV.

Article Snippet: Other expression plasmids used: pCAS-α-syn-GFP (a kind gift from Dr Oren Levy, Columbia University), pDsRed2-Mito (Clontech; no. 632421), and GFP-Sec61-β (Addgene; no. 15108).

Techniques: Labeling, Expressing, Quantitation Assay

Journal: The Journal of Neuroscience

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

doi: 10.1523/JNEUROSCI.2507-13.2014

Figure Lengend Snippet: Relationship of WT α-syn expression to mitochondrial fragmentation. A, M17 cells stably transfected with an EV plasmid or with the indicated plasmids expressing α-syn species were transiently cotransfected with plasmids expressing pDsRed2-Mito (red) and WT α-syn-GFP (green) and visualized by confocal microscopy. Cells were inspected visually and scored for whether they contained predominantly tubular or predominantly fragmented mitochondria (red). Note that, compared with Figure 7, the fragmented phenotype in the α-syn mutant cells is ameliorated by the expression of WT α-syn-GFP. B, Quantitation of percentage fragmented cells shown in A. *Significant difference versus corresponding untransfected cells (p < 0.05).

Article Snippet: Other expression plasmids used: pCAS-α-syn-GFP (a kind gift from Dr Oren Levy, Columbia University), pDsRed2-Mito (Clontech; no. 632421), and GFP-Sec61-β (Addgene; no. 15108).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Confocal Microscopy, Mutagenesis, Quantitation Assay

Journal: Oncotarget

Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells

doi: 10.18632/oncotarget.25415

Figure Lengend Snippet: (A) SUM159PT cells were transfected either with pCMV6-SNCG-GFP or pCMV6-A-GFP vector as a control (CTL) and GFP-positive cells were sorted by FACS. Representative photographs of SUM-CTL-GFP (left panels) and SUM-SNCG-GFP (right panels) cells expressing GFP (green) and stained with DAPI nuclear stain (blue); scale bar = 10 μm. (B) SNCG expression was analyzed by qRT-PCR using total RNA from SUM-CTL-GFP and SUM-SNCG-GFP (left panel). Levels of SNCG expression were normalized to those of RPLP0 internal control. Graph shows fold enrichment over SUM-CTL-GFP cells. Data represent mean values of two independent experiments performed in triplicate. Representative immunoblot analysis (n=3) was performed on whole cell lysate for SNCG-GFP fusion protein expression using anti β-actin antibodies as a loading control (right panel). (C) SUM-SNCG-GFP cells showed significant decreased apoptosis after radiation treatment (12 Gy) compared to SUM-CTL-GFP cells. Flow cytometry was done to measure apoptotic and necrotic cells. Histogram shows the percentage of apoptotic cells 72 hours after irradiation (left panel). Data represent mean values ± standard error of the mean of four independent experiments. Representative experiment of flow cytometry analysis (n=4) shows the percentage of Annexin-V and propidium iodide staining of SUM-CTL-GFP and SUM-SNCG-GFP cells irradiated or not at a dose of 12 Gy (right panel). (D) SUM-SNCG-GFP cells showed significant increased clonogenic potential after irradiation at 4 Gy compared to SUM-CTL-GFP cells. Clonogenic cell survival assay was performed; curves and bar graph show the percentage of survival after irradiation (left panel). Representative pictures of 2-week-old colonies after fixation and crystal violet staining (right panel). Data represent mean values ± standard error of the mean of three independent experiments. ** = P value < 0.01; * = P value < 0.05 ; ns = not significant.

Article Snippet: To establish stable cell lines expressing SNCG, SUM cells were transfected with a pCMV6-SNCG-GFP plasmid DNA (Origene, RG204173) or with the corresponding empty vector pCMV6-A-GFP plasmid DNA (Origene, PS100010) as a control.

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Quantitative RT-PCR, Western Blot, Flow Cytometry, Irradiation, Clonogenic Cell Survival Assay

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: Sphingolipids accumulating in GD promote the acceleration of α-synuclein aggregation into different pathologic species. A, Spatial topography of GlcCer metabolism in GD with reference to subcellular compartmentalization of enzymes and sphingolipids. Red upward arrows indicate upstream and downstream sphingolipids that accumulate peripherally due to GBA deficiency. Green zone represents lysosome. B, Summary of circular dichroism results obtained by incubating α-synuclein with the indicated lipids for 5 d. For all circular dichroism data, experimental lipids were added at 34.5 μm and α-synuclein added at 13.8 μm [(total lipid PC + experimental lipid liposome): protein = 10:1]. C, Circular dichroism spectra of WT α-synuclein at day 0. Unfolded proteins have minima at 195 nm, whereas α-helical conformations have minima at 205 and 222 nm. D, Circular dichroism spectra of WT α-synuclein at day 5. Proteins with β-sheeted conformations have minima at 217 nm. E, Time course of β-sheet conformation acquisition of WT α-synuclein in presence of GD sphingolipids. The percentage β-sheet conformation was calculated using DichroWeb. p values relative to PC for GlcCer = 0.005, GlcSph = 0.016, Sph = 0.024, S1P = 0.028, and Cer = 0.353. F, Time course of α-helical conformation acquisition in the presence of GM1 and BMP. p values relative to PC for BMP = 0.003, GM1 = 6.778 × 10−5. G, Time course of β-sheet conformation acquisition of mutant A53T α-synuclein in presence of same sphingolipids. p values relative to PC for GlcCer = 0.320, GlcSph = 0.003, Sph = 0.002, S1P = 0.049, Cer = 0.262. F, Time course of β-sheet conformation acquisition of mutant A30P α-synuclein in presence of same sphingolipids. p values relative to PC for GlcCer = 0.369, GlcSph = 0.012, Sph = 0.003, S1P = 8.0319 × 10−4, Cer = 0.481. E–H, Data are mean ± SEM. n = 3 experiments per sample, *p < 0.05 versus PC (one-tailed Student's t test). **p < 0.01 versus PC (one-tailed Student's t test). ***p < 0.001 versus PC (one-tailed Student's t test).

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques: Mutagenesis, One-tailed Test

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: GlcSph accumulates early in mouse brain: A, GlcCer; B, GlcSph; C, Sph; D, S1P levels in GD/PD brains. N = 2–6 per genotypes, except GbaKO/KO (N = 1); age of mice = 2–3 months. p value relative to WT/WT for GlcSph levels: L444P/WT = 0.234, N370S/WT = 0.313, KO/WT = 0.608, L444P/KO = 3.350 × 10−4, N370S/KO = 0.002. *p < 0.05. **p < 0.01. ***p < 0.001. E, AFM images of α-synuclein incubated with 0.1, 1, and 10 μm GlcSph with PC control on day 0 and 7. Scale bars, 1 μm. F, Percentage β-sheeted content of α-synuclein in the presence of 0.1, 1, and 10 μm GlcSph on day 0 and day 7. N = 3 for all samples. p value relative to PC: GlcSph-0.1 = 0.243, GlcSph-1 = 0.472, GlcSph-10 = 0.004. **p < 0.01 (two-tailed Student's t test). G, Subcellular fractionation showing lysosomes on the top fraction (F1) and mitochondria on the bottom fraction between 27% and 23% density interfaces (F2). Two smaller bands are seen between F1 and F2 (not taken). H, Western blot shows enrichment of lysosomes in F1. Cathepsin D, ATP6V1a, and LAMP1 were used as lysosomal markers; Cathepsin D and ATP6V1a can be seen in the homogenate, supernatant, and lysosomal fraction, but not in the mitochondrial fraction. LAMP1 can be seen predominantly in the lysosomal fraction. I, Relative protein levels in lysosomal fractions were determined by BCA. GBA KO lysosomes (11.38 mg/ml) were found to have almost 3 times as much protein as WT lysosomes (4.27 mg/ml), a likely indicator of expected lysosomal enlargement and dysfunction in the GBA KO mice. p value /KO relative to WT = 0.031. *p < 0.05 (two-tailed Student's t test). J, Dot blot shows relative GlcCer levels using a GlcCer antibody (Glycobiotech). Blotted GlcCer lipid shows that the dot blot method is robust and quantitative, with a standard curve with R2 = 0.99. K, GlcCer levels from brain homogenate, lysosomal fraction, and mitochondrial fraction were analyzed using the dot blot method in J. No difference between WT and GBA KO was found in any of the fractions, including total brain homogenates confirming the lipidomic results in A. However, there was a clear enrichment of GlcCer in the mitochondrial fraction relative to the others.

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques: Incubation, Two Tailed Test, Fractionation, Western Blot, Dot Blot

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: Oligomeric GlcSph and Sph α-synuclein species promote α-synuclein seeding. A, Intracellular α-synuclein aggregation assay in HEK293T cells, in which α-synuclein species aggregated in the presence of various lipids were added to cell media with a bioporter. Arrow indicates aggregated α-synuclein-GFP. Quantification shown for percentage of HEK293T cells with intracellular aggregates when templated with GD-related sphingolipid preformed α-synuclein species. N = 3 experiments with ≥241 cells per condition were assayed. p values relative to PC: Cer = 1.000, GM1 = 0.423, GlcCer = 0.301, GlcSph = 0.014, Sph = 4.900 × 10−4, S1P = 0.298. B, Intracellular α-synuclein aggregation assay as performed in human neurons. Quantification of relative aggregation, higher molecular weight species normalized to monomeric α-synuclein. N = 2 experiments. p values relative to PC: Cer = 0.894, GM1 = 0.544, GlcCer = 0.910, GlcSph = 0.021, Sph = 5.500 × 10−4, S1P = 0.867. *p < 0.05 (two-tailed Student's t test). ***p < 0.001 (two-tailed Student's t test).

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques: Molecular Weight, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: GD/PD mice exhibit α-synuclein pathology and GlcCer accumulation. A, Histograms of age of death of SNCATg mice (N = 12), heterozygous Gba /WTSNCATg (N = 36), and homozygous Gba /KOSNCATg (N = 18). Gaussian curve fitted to SNCATg histogram superimposed over all three histograms, showing differences in distributions; 1, 2, and 3 SDs from the mean shown through progressively darker shading under Gaussian curve. B, Quantification of GlcCer levels in the CA3 region of hippocampus in homozygous Gba /KOSNCATg brain of early death and anticipated death mice (N = 3 mice per group). N and p values relative to anticipated death: anticipated death: N = 3; early death: N = 3, p = 0.013. C, Quantification of GlcCer levels in the CA3 region of hippocampus in GD/PD mice cohorts. Values normalized to 1 for GbaWT/WT. N and p values relative to WT/WT: WT/WT: N = 3; SNCATg: N = 3, p = 0.624; /WT SNCATg: N = 4, p = 0.783; /KO SNCATg: N = 3, p = 0.010. D, Relative Ser129 phosphorylated α-synuclein levels in the CA3 region of the hippocampus in the same brains. N and p values relative to WT/WT: WT/WT: N = 3; SNCATg: N = 3, p = 0.010; /WT SNCATg: N = 4, p = 0.036; /KO SNCATg: N = 3, p = 0.010. E, Correlation of GlcCer and Ser129 phosphorylation α-synuclein levels across the different genotypes. r2 = 0.827, Pearson's product moment-correlation p = 1.618 × 10−5. N = 3 or 4 per genotype; age of mice = 8–11 months. F, Representative brain sections stained with antibodies to GlcCer and Ser129 phosphorylated α-synuclein. Scale bar, 250 μm. G, Western blot of soluble fraction of mouse brain homogenate following differential detergent extract in age-matched GbaWT/WT, SNCATg, and Gba/KOSNCATg exhibiting early death, probing for phosphorylated α-synuclein S129. H, Quantification of G, focusing on expected bands at 16 and 37 kDa. 16 kDa: N and p values versus WT/WT: SNCATg: N = 3, p = 4.120 × 10−4; /KO SNCATg: N = 3, p = 0.008. 37 kDa: N and p values versus WT/WT: SNCATg: N = 3, p = 0.090; /KO SNCATg: N = 3, p = 0.002. I, Dot blot of soluble fraction of mouse brain homogenate following differential detergent extract in age-matched GbaWT/WT, SNCATg, and Gba/KOSNCATg exhibiting early death, using anti-oligomer antibody A11. J, Quantification of ELISA assay probing for the presence of aggregated α-synuclein in total brain homogenate from age-matched GbaWT/WT, SNCATg, and Gba/KOSNCATg exhibiting early death. N and p values relative to WT/WT: WT/WT: N = 3; SNCATg: N = 3, p = 0.016; /KO SNCATg: N = 3, p = 0.014; p values versus SNCATg: /KO SNCATg = 0.034. B, C, Two-tailed Student's t test. D, H, J, One-tailed Student's t test. *p < 0.05. **p < 0.01.

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques: Staining, Western Blot, Dot Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, One-tailed Test

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: GlcSph and Sph promote oligomeric α-synuclein aggregates. A, Electron microscopy images of day 5 circular dichroism samples. Left panels, Images of monomeric and aggregated α-synuclein are shown for reference. Scale bar, 50 nm. B, AFM images of α-synuclein aggregated with Gaucher sphingolipids. Scale bar, 500 nm. Height of images = 50 nm. N = 3 independent experiments. C, Frequency distribution of size of α-synuclein species formed by aggregation in vitro as analyzed by AFM. Control conditions are in the absence of lipids, but aggregated for longer durations. The GD sphingolipids used are denoted. There are three images/condition. D, Median length of α-synuclein species formed.

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques: Electron Microscopy, In Vitro

Journal: The Journal of Neuroscience

Article Title: Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

doi: 10.1523/JNEUROSCI.1525-17.2017

Figure Lengend Snippet: Model of how GD-related sphingolipids impact α-synuclein pathology. Deletion or GD mutations in GBA leads to accumulation of GlcSph in the cytosol of neurons. GlcSph directly interacts with α-synuclein to promote its aggregation into distinct pathogenic oligomeric species. These pathogenic species further template intracellular α-synuclein aggregation and may have the capacity to spread to neighboring neurons. With age, GlcCer also accumulates; and there is a decrement of Gba2 and lysosomal enzymes, exacerbating α-synuclein pathology and proteostasis, leading to death of neurons.

Article Snippet: HEK293T cells were transfected with human α-synuclein-GFP (AddGene 40822) for 48 h using GenePORTER transfection reagent (Genlantis) in a 6-well plate.

Techniques:

Journal: eLife

Article Title: Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

doi: 10.7554/eLife.50034

Figure Lengend Snippet: ( A ) Confocal fluorescence microscopy of cultured rat hippocampal neurons co-transfected with plasmids encoding GFP-α-synuclein A53T mutant, mCherry transfection control, rat Birc6 shRNA-mCherry, and/or shRNA-resistant FLAG-tagged human BIRC6 (rescue), as indicated in the figure. Transfections were performed at day-in-vitro 3 (DIV3) and neurons were fixed for immunofluorescence microscopy at DIV7. Single-channel images are shown in inverted grayscale. Scale bar: 100 μm. Insets on the left and right columns are 7-fold magnified views from the axons in the boxed area. The inset in the middle bottom row is a 2.2-fold magnified view of FLAG-BIRC6 expression in the boxed area. ( B ) Magnified and straightened axons from control, Birc6-KD and FLAG-BIRC6-rescue neurons shown in A. Arrowheads indicate α-synuclein aggregates in the axon. ( C ) Quantification of the number of α-synuclein puncta (i.e., aggregates) per 100 μm of axon. Values are the mean ± SEM from 25 neurons from three independent experiments. The indicated p -values were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. ( D ) Cultured rat hippocampal neurons were transfected as in A. Neurons were extracted in Triton X-100 buffer and centrifuged. Supernatants were collected as the Triton-soluble fraction, while the pellets were resuspended in 5% SDS buffer as the Triton-insoluble fraction. Fractions were analyzed by SDS-PAGE and immunoblotting with antibodies to GFP (to detect GFP-α-synuclein) and β-tubulin (loading control). The positions of molecular mass markers (in kDa) are indicated on the left. ( E ) Quantification of the ratio of Triton-insoluble and -soluble GFP-α-synuclein. The value in control-shRNA–transfected neurons was set at 1. Values are the mean ± SEM from three independent experiments such as that in panel D. The indicated p -values were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. ( F ) Schematic representation of the role of UBA6 and BIRC6 in LC3 ubiquitination and targeting for degradation, decreasing the amount of LC3 that can be modified with PE for its function in autophagy.

Article Snippet: Recombinant DNA , GFP-α-synuclein A53T , Addgene; gift from David Rubinsztein , 40823 , .

Techniques: Fluorescence, Microscopy, Cell Culture, Transfection, Mutagenesis, Control, shRNA, In Vitro, Immunofluorescence, Expressing, SDS Page, Western Blot, Ubiquitin Proteomics, Modification

Journal: eLife

Article Title: Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

doi: 10.7554/eLife.50034

Figure Lengend Snippet: ( A ) Rat hippocampal neurons in primary culture were cotransfected with plasmids encoding GFP-α-synuclein A53T mutant and HA-Ub or mCherry-LC3B as indicated. Transfections were performed at day-in-vitro 3 (DIV3), and neurons were fixed for immunofluorescence microscopy at DIV7. Single-channel images are shown in inverted grayscale (left panels). Scale bar: 100 μm. Axons indicated by arrowheads were straightened and enlarged for better appreciation of protein colocalization (right panels). Merged images show that α-synuclein aggregates colocalize with Ub, SQSTM1 and LC3B, indicating that α-synuclein aggregates are ubiquitinated and recognized for selective autophagy. ( B ) Alignment of the shRNA-target sequence of genes encoding rat Birc6 and human BIRC6. The human BIRC6 gene has a two-nucleotide mismatch as compared to the shRNA designed for rat Birc6. ( C ) BIRC6-H2R (human to rat) plasmid was generated by substituting the shRNA-targeting site in human BIRC6 with the homologous rat Birc6 sequence. H4 cells were transfected with plasmids encoding BIRC6 or BIRC6-H2R together with the plasmid encoding Birc6 shRNA. Cells were lysed in 1xLDS sample buffer and analyzed by immunoblotting with antibodies to the indicated proteins. Notice the decrease in BIRC6-H2R levels by Birc6 shRNA, verifying that the Birc6 shRNA was able to silence the expression of rat Birc6 gene. The positions of molecular mass markers (in kDa) are indicated on the left.

Article Snippet: Recombinant DNA , GFP-α-synuclein A53T , Addgene; gift from David Rubinsztein , 40823 , .

Techniques: Mutagenesis, Transfection, In Vitro, Immunofluorescence, Microscopy, shRNA, Sequencing, Plasmid Preparation, Generated, Western Blot, Expressing

Journal: eLife

Article Title: Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

doi: 10.7554/eLife.50034

Figure Lengend Snippet: ( A ) Confocal fluorescence microscopy of cultured rat hippocampal neurons cotransfected at DIV3 with plasmids encoding GFP-α-synuclein A53T mutant, control shRNA-mCherry or rat Birc6 shRNA-mCherry as indicated in the figure. At DIV6, Birc6 shRNA-mCherry transfected neurons were incubated with 10 nM bafilomycin A 1 for 16 hr. Neurons were fixed for immunofluorescence assay at DIV7. Single-channel images are shown in inverted grayscale. Scale bar: 100 μm. Insets show magnified views of the axons indicated by arrowheads to better appreciation of α-synuclein aggregates. ( B ) Magnified and straightened axons from control, Birc6-KD and Birc6-KD–bafilomycin A 1 neurons shown in A. Arrowheads indicate mutant α-synuclein aggregates in the axon. ( C ) Quantification of the number of α-synuclein aggregates per 100 μm of axon. Values are the mean ± SEM from 25 neurons from three independent experiments. The indicated p -values were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. Notice that Birc6 KD decreased GFP-α-synuclein aggregate formation and that bafilomycin A 1 treatment reversed this effect of Birc6 KD.

Article Snippet: Recombinant DNA , GFP-α-synuclein A53T , Addgene; gift from David Rubinsztein , 40823 , .

Techniques: Fluorescence, Microscopy, Cell Culture, Mutagenesis, Control, shRNA, Transfection, Incubation, Immunofluorescence

Journal: eLife

Article Title: Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

doi: 10.7554/eLife.50034

Figure Lengend Snippet:

Article Snippet: Recombinant DNA , GFP-α-synuclein A53T , Addgene; gift from David Rubinsztein , 40823 , .

Techniques: FLAG-tag, Recombinant, Sequencing, Mutagenesis, CRISPR, Software