gfp rfp lc3bii  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Alpha-synuclein fragments trigger distinct aggregation pathways

doi: 10.1038/s41419-020-2285-7

Figure Lengend Snippet: a Experimental design. LUHMES cells, grown in differentiation medium from plating onward, were transduced with GFP or αSyn adenoviral vectors (AV) two days after plating. Viruses were removed 24 h after transduction. Cells and conditioned medium (CM) were harvested at the indicated readout times. DIV: days in vitro. DPT: days post-transduction. b αSyn-mediated toxicity. Cellular toxicity was monitored by lactate dehydrogenase (LDH) activity in the CM at the indicated readout times. Cells were either left untreated (Ctrl), challenged with a control AV expressing green fluorescent protein (GFP) or an AV expressing wild type αSyn. At DIV8, αSyn overexpression induced a considerably high toxicity, whilst GFP overexpression induced a mild toxicity, compared to untreated cells. LDH release values were related to a cell lysis positive control representing 100%. Data are presented as mean + standard error of the mean (SEM) from at least 3 biological repeats. ns: not significant, * p < 0.05, ** p < 0.005, *** p < 0.001; two-way ANOVA with Tukey’s post hoc test. c αSyn-overexpression in cell homogenates. Cell lysates were analysed by Western blot at the indicated readout times. αSyn overexpression levels were stable between DIV4 and DIV8. Aggregation of αSyn is visible in overexpressing cells, but not in untreated control or GFP overexpression conditions. αSyn bands smaller than 15 kDa were not detected, indicating absence of fragmented αSyn. f: fragments; m: monomer; o: oligomer; *: unspecific band. Actin was used as loading control. d αSyn in CM. Western blot analysis of αSyn in CM at the indicated times reveals the presence of several αSyn species at DIV6 and DIV8. An oligomer band appears at 37 kDa (o), a monomer band at 15 kDa (m) and several fragmented αSyn bands ranging from 13 to 6 kDa (f). CM of untreated and GFP overexpressing cells did not contain detectable levels of αSyn. Unconditioned medium (Med.) was used as control for unspecific bands (*). GAPDH was used as control for cytoplasmic content in the CM.

Article Snippet: Adenoviral vectors expressing human wild-type alpha synuclein (αSyn) or green fluorescent protein (GFP) under a cytomegalovirus promoter (BioFocus DPI, Leiden, Netherlands) were added to LUHMES cells 48 h after start of differentiation with a multiplicity of infection (MOI) of 2, as previously described , .

Techniques: Transduction, In Vitro, Activity Assay, Control, Expressing, Over Expression, Lysis, Positive Control, Western 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: 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: