human cfpstim1  (Addgene inc)

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GCase protein level and activity in patient-derived fibroblasts. ( A ) Immunoblot and quantification for the GCase protein level in fibroblasts normalized to WT/WT control fibroblasts as shown graphically as mean with SEM ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.001; n = 3). Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ( B ) Immunoblot and quantification for the GCase protein level in adult and young control fibroblasts normalized to WT/WT control fibroblasts ( * * * * P < 0.0001; n = 3). Three technical repeats. ( C ) The expression of GBA mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, GBA expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean and error bars show the SEM. GCase activity assay with M-Glu was performed at ( D ) pH 5.4 with NaT and ( E ) pH 4.5 on fibroblast cell lysates. All data normalized to WT/WT control fibroblasts. GCase activity in in adult and young control fibroblasts at ( F ) pH 5.4 with NaT and (G) pH 4.5. ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4; ns, not significant). All data normalized to WT/WT control fibroblasts. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. Statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at: https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Activity Assay, Derivative Assay, Western Blot, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lysosomal content and function in patient-derived fibroblasts. ( A ) LAMP1 levels were measured via western blot and quantified to assess the overall endo-lysosomal content of the fibroblasts. Data quantified and normalized to WT/WT fibroblast controls. Three technical repeats. ( B ) LAMP1 levels measured via western blot and compared with young control fibroblasts. Lysosomal function in patient-derived fibroblasts. Three technical repeats. ( C ) β-Galactosidase and ( D ) β-hexosaminidase were measured at pH 4.1 to assess the overall lysosomal function in fibroblasts. All data normalized to WT/WT control fibroblasts. Four technical repeats. The activities of lysosomal hydrolases, ( E ) β-galactosidase and ( F ) β-hexosaminidase were measured with young control fibroblasts. Four technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. For the analysis of young controls, WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. The statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: ER retention and ER stress in patient-derived fibroblasts. ( A ) Fibroblast cell lysates (20 μg of WT, E326K and N370S mutants and 70 μg of L444P mutants) were treated with or without endoglycosidase-H (Endo H) and GCase protein species analysed by western blot. WT/WT cell lysate (20 μg) was treated with Peptide-N-Glycosidase F (PNGase F) as a positive control. Figure shows blots at long and short exposures. The two normal species of GCase detected in fibroblasts are indicated by arrows. An additional lower molecular weight band was observed, indicating ER retained GCase, in L444P/L444P fibroblasts following endo-H treatment (black asterisk). The WT/WT cell line treated with PNGase exhibited a lower molecular weight band, indicating a GCase species with no glycosylation (Δ). ( B ) Quantitative analysis of Endo H digestion displayed as the density of bands for ER resident GCase divided by the density of bands for total GCase protein, normalized to the β-actin band density. Data normalized to WT/WT that was set at 1. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ER stress was analysed by ( C ) quantifying the BiP protein level in fibroblasts. Results are normalized to WT/WT control fibroblasts. Three technical repeats. ( D ) The expression of CHOP mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, CHOP expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean with error bars showing the SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis ( * P < 0.05, * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Positive Control, Molecular Weight, Glycoproteomics, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GBA levels, lysosomal function and ER stress in SH-SY5Y cells overexpressing mutant GCase. ( A ) Quantification of GBA mRNA levels in stable SH-SY5Y cell lines, normalized to untransfected SH-SY5Y cells (set at 1). Three technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1. ( B ) Immunoblot and ( C ) quantification of GBA protein levels in stable SH-SY5Y cell lines. Data normalized to untransfected SH-SY5Y cells (set at 1). Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4). Four technical repeats. ( D ) GCase activity in nmole/hr/mg in stable SH-SY5Y cell lines measured at pH 5.4 with NaT and normalized to untransfected SH-SY5Y cells (set at 1) Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001; n = 4). The activities of lysosomal hydrolases, β-galactosidase and β-hexosaminidase were measured at pH 5.1 in SH-SY5Y stable cell lines. Four technical repeats. ( E ) β-galactosidase activity in nmole/0.5 h/mg in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. ( F ) β-Hexosaminidase activity in nmole/0.5 h/mg measured in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. Four technical repeats. ( G ) LAMP1 levels were measured via western blot and ( H ) quantified to assess the overall endo-lysosomal content of the SH-SY5Y cell lines. Immunoblot and quantification for LAMP1 protein level in SH-SY5Y stable cell lines. Data normalized to wild-type SH-SY5Y cells. Four technical repeats. ( I ) Immunoblot and ( J ) quantification of BiP protein level in SH-SY5Y clones. Data normalized to untransfected SH-SY5Y cells. Six technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis( * P < 0.05. * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Western Blot, Activity Assay, Stable Transfection, Clone Assay

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Soluble and insoluble alpha-synuclein levels in SH-SY5Y stable cell lines expressing mutant GBA. ( A ) Immunoblot of alpha-synuclein protein level in SH-SY5Y clones. ( B ) Quantification of alpha-synuclein immunoblotting in SH-SY5Y clones normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Six technical repeats. ( C ) Quantification of SNCA mRNA levels in SH-SY5Y stable clones normalized to wild-type clones ( n = 3). The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Two biological repeats and six technical repeats. TX-100 soluble and insoluble fractions (urea-SDS) were made from cells and analysed for alpha-synuclein by western blotting. HMW alpha-synuclein species (arrow) were detected in urea-SDS fractions. Monomeric alpha-synuclein was present in the TX-100 soluble fraction and some urea-SDS fractions. ( D ) Immunoblot for all SH-SY5Y cell lines over-expressing mutant GBA protein. Appropriate control cell lines were also analysed: ( E ) HAP1 alpha-synuclein knockout cells (KO), ( F ) untransfected SH-SY5Y cells (UT) and ( G ) GFP over-expressing SH-SY5Y cells (GFP). ( H ) Quantification of soluble and insoluble alpha-synuclein immunoblots. Data normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Ten technical repeats. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; n = 16). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Stable Transfection, Expressing, Mutagenesis, Western Blot, Clone Assay, Control, Knock-Out

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lipid droplets in GBA mutant cells. ( A ) Fibroblast lines harbouring the E326K mutations were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. Scale bar represents 20 μm. The n for each genotype per experiment was WT/WT n = 6; E326K/WT n = 1; E326K/E326K n = 1; >100 cells analysed per genotype. Quantification of lipid droplets in ( B ) control and E326K/+ fibroblasts and ( C) control and E326K/E326K fibroblasts displayed as the mean with error bars showing SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * * * P < 0.0001). ( D ) SH-SY5Y cells over-expressing mutant GCase protein were grown on coverslips and stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( E ) Quantification of lipid droplets per cell area shown as the mean with SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * P < 0.01; * * * * P < 0.0001). ( F ) SH-SY5Y cells over-expressing mutant GCase protein were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( G ) Quantification of lipid droplets per cell area displayed as mean with error bars showing SEM. Statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; * * * * P < 0.0001). Scale bar represents 20 μm. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Incubation, Staining, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GCase protein level and activity in patient-derived fibroblasts. ( A ) Immunoblot and quantification for the GCase protein level in fibroblasts normalized to WT/WT control fibroblasts as shown graphically as mean with SEM ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.001; n = 3). Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ( B ) Immunoblot and quantification for the GCase protein level in adult and young control fibroblasts normalized to WT/WT control fibroblasts ( * * * * P < 0.0001; n = 3). Three technical repeats. ( C ) The expression of GBA mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, GBA expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean and error bars show the SEM. GCase activity assay with M-Glu was performed at ( D ) pH 5.4 with NaT and ( E ) pH 4.5 on fibroblast cell lysates. All data normalized to WT/WT control fibroblasts. GCase activity in in adult and young control fibroblasts at ( F ) pH 5.4 with NaT and (G) pH 4.5. ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4; ns, not significant). All data normalized to WT/WT control fibroblasts. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. Statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at: https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Activity Assay, Derivative Assay, Western Blot, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lysosomal content and function in patient-derived fibroblasts. ( A ) LAMP1 levels were measured via western blot and quantified to assess the overall endo-lysosomal content of the fibroblasts. Data quantified and normalized to WT/WT fibroblast controls. Three technical repeats. ( B ) LAMP1 levels measured via western blot and compared with young control fibroblasts. Lysosomal function in patient-derived fibroblasts. Three technical repeats. ( C ) β-Galactosidase and ( D ) β-hexosaminidase were measured at pH 4.1 to assess the overall lysosomal function in fibroblasts. All data normalized to WT/WT control fibroblasts. Four technical repeats. The activities of lysosomal hydrolases, ( E ) β-galactosidase and ( F ) β-hexosaminidase were measured with young control fibroblasts. Four technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. For the analysis of young controls, WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. The statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: ER retention and ER stress in patient-derived fibroblasts. ( A ) Fibroblast cell lysates (20 μg of WT, E326K and N370S mutants and 70 μg of L444P mutants) were treated with or without endoglycosidase-H (Endo H) and GCase protein species analysed by western blot. WT/WT cell lysate (20 μg) was treated with Peptide-N-Glycosidase F (PNGase F) as a positive control. Figure shows blots at long and short exposures. The two normal species of GCase detected in fibroblasts are indicated by arrows. An additional lower molecular weight band was observed, indicating ER retained GCase, in L444P/L444P fibroblasts following endo-H treatment (black asterisk). The WT/WT cell line treated with PNGase exhibited a lower molecular weight band, indicating a GCase species with no glycosylation (Δ). ( B ) Quantitative analysis of Endo H digestion displayed as the density of bands for ER resident GCase divided by the density of bands for total GCase protein, normalized to the β-actin band density. Data normalized to WT/WT that was set at 1. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ER stress was analysed by ( C ) quantifying the BiP protein level in fibroblasts. Results are normalized to WT/WT control fibroblasts. Three technical repeats. ( D ) The expression of CHOP mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, CHOP expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean with error bars showing the SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis ( * P < 0.05, * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Positive Control, Molecular Weight, Glycoproteomics, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GBA levels, lysosomal function and ER stress in SH-SY5Y cells overexpressing mutant GCase. ( A ) Quantification of GBA mRNA levels in stable SH-SY5Y cell lines, normalized to untransfected SH-SY5Y cells (set at 1). Three technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1. ( B ) Immunoblot and ( C ) quantification of GBA protein levels in stable SH-SY5Y cell lines. Data normalized to untransfected SH-SY5Y cells (set at 1). Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4). Four technical repeats. ( D ) GCase activity in nmole/hr/mg in stable SH-SY5Y cell lines measured at pH 5.4 with NaT and normalized to untransfected SH-SY5Y cells (set at 1) Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001; n = 4). The activities of lysosomal hydrolases, β-galactosidase and β-hexosaminidase were measured at pH 5.1 in SH-SY5Y stable cell lines. Four technical repeats. ( E ) β-galactosidase activity in nmole/0.5 h/mg in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. ( F ) β-Hexosaminidase activity in nmole/0.5 h/mg measured in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. Four technical repeats. ( G ) LAMP1 levels were measured via western blot and ( H ) quantified to assess the overall endo-lysosomal content of the SH-SY5Y cell lines. Immunoblot and quantification for LAMP1 protein level in SH-SY5Y stable cell lines. Data normalized to wild-type SH-SY5Y cells. Four technical repeats. ( I ) Immunoblot and ( J ) quantification of BiP protein level in SH-SY5Y clones. Data normalized to untransfected SH-SY5Y cells. Six technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis( * P < 0.05. * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Western Blot, Activity Assay, Stable Transfection, Clone Assay

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Soluble and insoluble alpha-synuclein levels in SH-SY5Y stable cell lines expressing mutant GBA. ( A ) Immunoblot of alpha-synuclein protein level in SH-SY5Y clones. ( B ) Quantification of alpha-synuclein immunoblotting in SH-SY5Y clones normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Six technical repeats. ( C ) Quantification of SNCA mRNA levels in SH-SY5Y stable clones normalized to wild-type clones ( n = 3). The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Two biological repeats and six technical repeats. TX-100 soluble and insoluble fractions (urea-SDS) were made from cells and analysed for alpha-synuclein by western blotting. HMW alpha-synuclein species (arrow) were detected in urea-SDS fractions. Monomeric alpha-synuclein was present in the TX-100 soluble fraction and some urea-SDS fractions. ( D ) Immunoblot for all SH-SY5Y cell lines over-expressing mutant GBA protein. Appropriate control cell lines were also analysed: ( E ) HAP1 alpha-synuclein knockout cells (KO), ( F ) untransfected SH-SY5Y cells (UT) and ( G ) GFP over-expressing SH-SY5Y cells (GFP). ( H ) Quantification of soluble and insoluble alpha-synuclein immunoblots. Data normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Ten technical repeats. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; n = 16). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Stable Transfection, Expressing, Mutagenesis, Western Blot, Clone Assay, Control, Knock-Out

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lipid droplets in GBA mutant cells. ( A ) Fibroblast lines harbouring the E326K mutations were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. Scale bar represents 20 μm. The n for each genotype per experiment was WT/WT n = 6; E326K/WT n = 1; E326K/E326K n = 1; >100 cells analysed per genotype. Quantification of lipid droplets in ( B ) control and E326K/+ fibroblasts and ( C) control and E326K/E326K fibroblasts displayed as the mean with error bars showing SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * * * P < 0.0001). ( D ) SH-SY5Y cells over-expressing mutant GCase protein were grown on coverslips and stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( E ) Quantification of lipid droplets per cell area shown as the mean with SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * P < 0.01; * * * * P < 0.0001). ( F ) SH-SY5Y cells over-expressing mutant GCase protein were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( G ) Quantification of lipid droplets per cell area displayed as mean with error bars showing SEM. Statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; * * * * P < 0.0001). Scale bar represents 20 μm. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Incubation, Staining, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Proposed mechanisms underlying E326K GCase pathology. (1) E326K GCase is likely not retained in the ER and is trafficked to the lysosome. At the lysosome, GCase carries out its ‘moonlighting’ function of glucosylating cholesterol. E326K Gcase may have increased or decreased the ability to glucosylate cholesterol, which alters the properties of accumulated cholesterol. This cholesterol can interfere with lipid membranes, altering lipid composition and promoting alpha-synuclein aggregation. This cholesterol has altered properties so may be more prone to storage in to lipid droplets, leading to an accumulation of lipid droplets. Lipid droplets can act as a site to bind alpha-synuclein, and in high concentrations and under pathological conditions, alpha-synuclein may aggregate at lipid droplets. (2) The E326K GCase mutation may lead to dysfunctional mitochondria, potentially through impaired clearance. These dysfunctional mitochondria may have reduced ability to metabolize fatty acids, leading to the accumulation of FFA in the neuron. FFA are capable of interfering with lipid membranes, potentially promoting alpha-synuclein aggregation. FFA are also sequestered into lipid droplets, as the neuron attempts to protect the cell from lipotoxicity. (3) The E326K mutation may cause a shift in the mitochondria’s metabolism capacity, shifting away from glycolysis toward fatty acid oxidation to provide energy for the neuron. In order to meet the demand for fatty acids, the neuron may be more primed to synthesize fatty acids or take up external fatty acids. This may lead to an accumulation of FFA in the neuron, which can exert lipotoxic effects, accumulate in lipid droplets and may induce alpha-synuclein aggregation.

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GCase protein level and activity in patient-derived fibroblasts. ( A ) Immunoblot and quantification for the GCase protein level in fibroblasts normalized to WT/WT control fibroblasts as shown graphically as mean with SEM ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.001; n = 3). Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ( B ) Immunoblot and quantification for the GCase protein level in adult and young control fibroblasts normalized to WT/WT control fibroblasts ( * * * * P < 0.0001; n = 3). Three technical repeats. ( C ) The expression of GBA mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, GBA expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean and error bars show the SEM. GCase activity assay with M-Glu was performed at ( D ) pH 5.4 with NaT and ( E ) pH 4.5 on fibroblast cell lysates. All data normalized to WT/WT control fibroblasts. GCase activity in in adult and young control fibroblasts at ( F ) pH 5.4 with NaT and (G) pH 4.5. ( * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4; ns, not significant). All data normalized to WT/WT control fibroblasts. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. Statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at: https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Activity Assay, Derivative Assay, Western Blot, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lysosomal content and function in patient-derived fibroblasts. ( A ) LAMP1 levels were measured via western blot and quantified to assess the overall endo-lysosomal content of the fibroblasts. Data quantified and normalized to WT/WT fibroblast controls. Three technical repeats. ( B ) LAMP1 levels measured via western blot and compared with young control fibroblasts. Lysosomal function in patient-derived fibroblasts. Three technical repeats. ( C ) β-Galactosidase and ( D ) β-hexosaminidase were measured at pH 4.1 to assess the overall lysosomal function in fibroblasts. All data normalized to WT/WT control fibroblasts. Four technical repeats. The activities of lysosomal hydrolases, ( E ) β-galactosidase and ( F ) β-hexosaminidase were measured with young control fibroblasts. Four technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. For the analysis of young controls, WT/WT n = 2; young WT/WT n = 2; L444P/L444P n = 2. All graphs show the mean with error bars as the SEM. The statistical test used was one-away ANOVA with Tukey’s post hoc analysis. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: ER retention and ER stress in patient-derived fibroblasts. ( A ) Fibroblast cell lysates (20 μg of WT, E326K and N370S mutants and 70 μg of L444P mutants) were treated with or without endoglycosidase-H (Endo H) and GCase protein species analysed by western blot. WT/WT cell lysate (20 μg) was treated with Peptide-N-Glycosidase F (PNGase F) as a positive control. Figure shows blots at long and short exposures. The two normal species of GCase detected in fibroblasts are indicated by arrows. An additional lower molecular weight band was observed, indicating ER retained GCase, in L444P/L444P fibroblasts following endo-H treatment (black asterisk). The WT/WT cell line treated with PNGase exhibited a lower molecular weight band, indicating a GCase species with no glycosylation (Δ). ( B ) Quantitative analysis of Endo H digestion displayed as the density of bands for ER resident GCase divided by the density of bands for total GCase protein, normalized to the β-actin band density. Data normalized to WT/WT that was set at 1. Three technical repeats. The n for each genotype per experiment was WT/WT n = 2; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. ER stress was analysed by ( C ) quantifying the BiP protein level in fibroblasts. Results are normalized to WT/WT control fibroblasts. Three technical repeats. ( D ) The expression of CHOP mRNA levels in patient fibroblasts was quantified and normalized to WT/WT controls. For each experiment, two biological replicates were used for each cell line. For quantification, CHOP expression for each cell line was calculated, pooled and averaged for each genotype. Three technical repeats. The n for each genotype per experiment was WT/WT n = 3; E326K/WT n = 1; E326K/E326K n = 1; L444P/L444P n = 2; N370S/N370S n = 2. Graphs show the mean with error bars showing the SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis ( * P < 0.05, * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Derivative Assay, Western Blot, Positive Control, Molecular Weight, Glycoproteomics, Control, Expressing

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: GBA levels, lysosomal function and ER stress in SH-SY5Y cells overexpressing mutant GCase. ( A ) Quantification of GBA mRNA levels in stable SH-SY5Y cell lines, normalized to untransfected SH-SY5Y cells (set at 1). Three technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1. ( B ) Immunoblot and ( C ) quantification of GBA protein levels in stable SH-SY5Y cell lines. Data normalized to untransfected SH-SY5Y cells (set at 1). Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001; n = 4). Four technical repeats. ( D ) GCase activity in nmole/hr/mg in stable SH-SY5Y cell lines measured at pH 5.4 with NaT and normalized to untransfected SH-SY5Y cells (set at 1) Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05, * * P < 0.01, * * * P < 0.001; n = 4). The activities of lysosomal hydrolases, β-galactosidase and β-hexosaminidase were measured at pH 5.1 in SH-SY5Y stable cell lines. Four technical repeats. ( E ) β-galactosidase activity in nmole/0.5 h/mg in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. ( F ) β-Hexosaminidase activity in nmole/0.5 h/mg measured in undifferentiated SH-SY5Y clones normalized to untransfected SH-SY5Y cells. Four technical repeats. ( G ) LAMP1 levels were measured via western blot and ( H ) quantified to assess the overall endo-lysosomal content of the SH-SY5Y cell lines. Immunoblot and quantification for LAMP1 protein level in SH-SY5Y stable cell lines. Data normalized to wild-type SH-SY5Y cells. Four technical repeats. ( I ) Immunoblot and ( J ) quantification of BiP protein level in SH-SY5Y clones. Data normalized to untransfected SH-SY5Y cells. Six technical repeats. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis( * P < 0.05. * * P < 0.01; n = 3). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Western Blot, Activity Assay, Stable Transfection, Clone Assay

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Soluble and insoluble alpha-synuclein levels in SH-SY5Y stable cell lines expressing mutant GBA. ( A ) Immunoblot of alpha-synuclein protein level in SH-SY5Y clones. ( B ) Quantification of alpha-synuclein immunoblotting in SH-SY5Y clones normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Six technical repeats. ( C ) Quantification of SNCA mRNA levels in SH-SY5Y stable clones normalized to wild-type clones ( n = 3). The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Two biological repeats and six technical repeats. TX-100 soluble and insoluble fractions (urea-SDS) were made from cells and analysed for alpha-synuclein by western blotting. HMW alpha-synuclein species (arrow) were detected in urea-SDS fractions. Monomeric alpha-synuclein was present in the TX-100 soluble fraction and some urea-SDS fractions. ( D ) Immunoblot for all SH-SY5Y cell lines over-expressing mutant GBA protein. Appropriate control cell lines were also analysed: ( E ) HAP1 alpha-synuclein knockout cells (KO), ( F ) untransfected SH-SY5Y cells (UT) and ( G ) GFP over-expressing SH-SY5Y cells (GFP). ( H ) Quantification of soluble and insoluble alpha-synuclein immunoblots. Data normalized to wild-type clones. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. Ten technical repeats. Graphs show the mean with SEM. The statistical test used was one-way ANOVA with Tukey’s post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; n = 16). Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Stable Transfection, Expressing, Mutagenesis, Western Blot, Clone Assay, Control, Knock-Out

Journal: Human Molecular Genetics

Article Title: The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

doi: 10.1093/hmg/ddac233

Figure Lengend Snippet: Lipid droplets in GBA mutant cells. ( A ) Fibroblast lines harbouring the E326K mutations were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. Scale bar represents 20 μm. The n for each genotype per experiment was WT/WT n = 6; E326K/WT n = 1; E326K/E326K n = 1; >100 cells analysed per genotype. Quantification of lipid droplets in ( B ) control and E326K/+ fibroblasts and ( C) control and E326K/E326K fibroblasts displayed as the mean with error bars showing SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * * * P < 0.0001). ( D ) SH-SY5Y cells over-expressing mutant GCase protein were grown on coverslips and stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( E ) Quantification of lipid droplets per cell area shown as the mean with SEM. The statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * * P < 0.01; * * * * P < 0.0001). ( F ) SH-SY5Y cells over-expressing mutant GCase protein were starved in Opti-MEM overnight and incubated with and without 100 μM OA for 5 h. Cells were stained with lipophilic fluorescent probe BODIPY 493/503. Lipid droplets represent punctate structures. Three representative images from each genotype shown (−) denotes untreated and (+) denotes treated with OA. All cells were counted in the images and the number of lipid droplets counted and normalized to cell area using ImageJ. The n for each genotype per experiment was wild type n = 2; E326K n = 2; L444P n = 2; N370S n = 2; untransfected n = 1; GFP n = 1. > 50 cells analysed per genotype. ( G ) Quantification of lipid droplets per cell area displayed as mean with error bars showing SEM. Statistical test used was one-way ANOVA with Tukey post hoc analysis or two-way ANOVA with Tukey’s post hoc analysis ( * P < 0.05; * * * * P < 0.0001). Scale bar represents 20 μm. Raw data can be found at https://doi.org/10.5281/zenodo.6985167 .

Article Snippet: SH-SY5Y cells were transfected with a pcDNATM3.1 (+) mammalian expression vector (ThermoFisher, Cat#V79020) containing wild type (WT GBA pcDNA3.1; Addgene plasmid #188580; RRID: Addgene_188 580), E326K (E326K GBA pcDNA3.1; Addgene plasmid #188581; RRID: Addgene_188 581), L444P (L444P GBA pcDNA3.1; Addgene plasmid #188582; RRID: Addgene_188 582) or N370S (N370S GBA pcDNA3.1; Addgene plasmid #188583; RRID: Addgene_188 583), GBA cDNA.

Techniques: Mutagenesis, Incubation, Staining, Control, Expressing