gba1-e326k mutation  (DWK Life Sciences)

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Representative micrographs of data quantified in B from WT, GBA1 -p.E326K KI, GBA1 -p.L444P KI, and GBA1 KO cells depicting LysoFQ-GBA probe fluorescence (green). Nuclei stained with DAPI (blue); scale bar is 10 µm. B WT, GBA1 -p.E326K KI, GBA1 -p.L444P KI, and GBA1 KO HEK293T cells were stained with LysoFQ-GBA probe to measure lysosomal GCase activity, and the mean GCase activity for each clone is quantified as the sum LysoFQ-GBA spot intensity per nuclei, normalized to the mean of all WT cells. n = 3 independent experiments. C Representative immunoblot of whole cell lysates from HEK293T cells of indicated genotype assessed with antibodies against GCase and actin as a loading control. D Quantification of total cellular GCase enzyme levels, measured via immunoblot & normalized to the levels of actin and the mean signal from WT cells. n = 3 independent experiments. E Representative immunoblot of Lyso-IP samples from HEK293T cells (of indicated genotype) expressing TMEM192-HA x3 tag. Immunoblots were probed using antibodies against GCase, HA, NPC2, and LIMP2. F Quantification of lysosomal GCase levels (normalized per sample to HA band intensity), measured via immunoblotting of Lyso-IP fraction and normalized to the mean of all WT samples. n = 4 independent experiments. G Representative immunoblot of total cell lysates from HEK293T cells of indicated genotype, treated ± Endoglycosidase H (EndoH), and probed using antibodies against GCase and actin as a loading control. H Quantification of the EndoH-resistant GCase band intensity normalized to the total GCase band intensity, from EndoH-treated cell lysates from HEK293T cells of indicated genotype. n = 4 independent experiments. I Representative immuoblots from Strep-Tactin pulldowns performed on lysates from GBA1 KO HEK293T cells transfected with StrepII-tagged GBA1 cDNA for WT GBA1 and GBA1 -p.E326K. Immunoblots were probed using antibodies against LIMP2, GCase and Strep. J Quantification of LIMP2:GCase band intensity ratios, normalized to the WT GBA1/pH 7.3 lysis condition within each replicate, from pulldown fraction of experiment shown in I. n = 4 independent experiments. All bar graphs depict mean ± SEM with individual points representative of data from a single experimental replicate. Unless otherwise noted, all statistics were performed on genotype-level comparisons using one-way ANOVA with Dunnett’s multiple comparison test, where ** = p < 0.0021, *** = p < 0.0002, **** = p < 0.0001.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Fluorescence, Staining, Activity Assay, Western Blot, Control, Expressing, Transfection, Lysis, Comparison

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Enzymatic activity of purified recombinant GCase (WT or E326K) against the substrate 4-methylumbelliferyl b-glucopyranoside (4-MUG), at an enzyme concentration of 2 nM. Curves depict data fits to Michaelis-Menten kinetics model (top). B Selected enzymatic parameters, derived from Michaelis-Menten kinetics shown in B . C Enzymatic activity of purified recombinant GCase (WT or E326K) against GlcCer in liposomes prepared with either neutral lipids (gray bars) or with 30% BMP (orange bars). Imiglucerase is colored in grey, recombinant WT GCase in blue and E326K in orange. n = 3 independent experiments. All bar graphs depict mean ± SEM with individual points representative of data from a single experimental replicate. D Overlay of size exclusion chromatography profiles of purified WT (blue) and E326K (orange) GCase. Samples were run over a Superdex S200 3.2/100 pre-equilibrated in 20mM sodium acetate pH 5.5 and 150mM NaCl and absorbance at 280nm was recorded. Early elution profile for E326K suggests that protein behaves as a multimer compared to WT GCase. Void volume is expected around 0.9mL confirming E326K is not a soluble aggregate. E Binding affinities measured by SPR for the LIMP2-GCase interaction at both neutral and acidic pHs, with LIMP2 lumenal domain immobilized on the chip. Values correspond to the average of 2 independent replicates, using either a 1:1 (top table) or biphasic (bottom table) binding model. F SEC-MALS analysis of the GCase E326K / LIMP2 complex is consistent with a 2:2 dimeric organization (observed molecular weight ∼ 200 kDa, expected MW for GCase ∼50kDa and LIMP2 extracellular domain ∼45kDa, not accounting for glycosylation). Absorbance trace is shown in blue, and apparent molecular weight is shown in purple. G CryoEM reconstruction of the E326K variant & LIMP2 lumenal domain complex at a resolution of 3.3 Å. Two orthogonal side views of an isosurface rendering of the complex are shown. LIMP2 N- and C-termini are indicated relative to the lysosomal membrane. H Cartoon representation of the E326K variant & LIMP2 complex structure (center). K326 in both E326K variant protomers is shown in red spheres. Overlay of Loop 1 in E326K variant & LIMP2 complex structure with helical (blue) and extended (green) conformations of Loop 1 observed in WT GCase at neutral pH (PDB: 2NT1, chains A and B) and (top left). Close-up view of E326K dimer interface showing that the interface consists primarily of hydrophobic interactions (upper right). Close-up view of interactions forming the E326K variant interface with LIMP2 (bottom left). Close-up view of the electrostatic interactions at the LIMP2 dimer interface (bottom right).

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Activity Assay, Purification, Recombinant, Concentration Assay, Derivative Assay, Liposomes, Size-exclusion Chromatography, Binding Assay, Molecular Weight, Glycoproteomics, Variant Assay, Membrane

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Crystal structure of the GCase E326K variant dimer at 3.1 Å. Overlay of the E326K variant dimer observed in the crystal structure of E326K alone (gold) and the cryoEM structure of the E326K variant & LIMP2 complex (green). B Close-up view of the interactions of residue 326 in GCase structures. In WT GCase (PDB:2NT1, blue) the carboxylic side chain of Glu326 engages in a salt bridge interaction with guanidium group of R329. This electrostatic interaction is lost in both the E326K variant crystal structure (gold) and the cryoEM structure of the E326K variant & LIMP2 complex (green). C Corresponding electron density (mesh representation) for panel B (contoured at 1σ for the WT and E326K crystal structures, and 8σ for the E326K/LIMP2 cryoEM structure), showing that the E326 and R329 sidechains are both well-ordered and well-resolved in WT GCase, while the K326 and R329 sidechains are more disordered in the E326K variant. D Size exclusion chromatography profiles of purified E326K+R329A (purple), E326K+R329E (light red), and E326K+R329A (violet) recombinant enzyme. Elution profiles for WT GCase and E326K variant are shown in blue and orange, respectively. Elution profiles of monomeric and dimeric proteins are indicated with text. E Representative immunoblot from Strep-Tactin pulldowns performed on lysates from GBA1 KO HEK293T cells transfected with StrepII-tagged GBA1 cDNA for WT GBA1 , GBA1 -p.E326K, and E326K+R329D/E/A variants. Immunoblots were probed using antibodies against LIMP2, GCase and Strep. F Quantification of LIMP2:GCase band intensity ratios, normalized to the WT GBA1 transfected condition within each replicate, from pulldown fraction of experiment shown in E . n = 3 independent experiments, statistics performed using one-way ANOVA with Dunnett’s multiple comparison test to WT GCase, where *** = p < 0.0002, **** = p < 0.0001. All bar graphs depict mean ± SEM with individual points representative of data from a single experimental replicate.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Variant Assay, Residue, Size-exclusion Chromatography, Purification, Recombinant, Western Blot, Transfection, Comparison

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Heatmaps depicting log 2 fold changes (from WT mean) in the abundance of all detected GCase substrates from whole-cell and Lyso-IP fractions taken from WT, GBA1 -p.E326K KI, GBA1 -p.L444P KI, and GBA1 KO HEK293T cells. B The fold change (over WT mean) of glucosylsphingosine (GlcSph) levels in Lyso-IP samples from HEK293T cells of the indicated genotype. C Schematic depicting the major lipids and key enzymes/protein co-factors in the lysosomal glycosphingolipid degradation pathway proximal to GCase. D Heatmaps showing the log 2 fold change (from WT mean) of glycosphingolipids (from pathway shown in c) in both whole-cell and Lyso-IP fractions taken from HEK293T cells of indicated genotype. Only lipids with a fold change of E326K vs WT that reached nominal significance (p < 0.1) in either the whole-cell or Lyso-IP fractions are included. E Fold change (over WT mean) of BMP(22:6/22:6) levels in Lyso-IP samples from HEK293T cells of the indicated genotype. F Fold change (over WT mean) of glucosylsphingosine levels in whole cell samples from HEK293T cells of the indicated genotype, treated with imiglucerase as indicated. G & H Fold change (over WT mean) of BMP(22:6/22:6) (G) or GB3(d18:1/18:0) (H) levels in whole cell samples from HEK293T cells of the indicated genotype, treated with imiglucerase as indicated. All bar graphs show mean ± SEM with individual points representative of data from a single experimental replicate. Data in A-B, and D-E are from n = 4 independent replicates. Data in F-H are from n = 5 independent replicates. For A and D, statistical tests were performed on genotype-level comparisons using robust linear model as described in methods and significance threshold was set at FDR adjusted P value < 0.05. All other statistical tests were performed on genotype-level comparisons using one-way ANOVA with Dunnett’s multiple comparison test (B and E), or Tukey’s multiple comparisons test (F-H), where * = p < 0.0332, *** = p < 0.0002, **** = p < 0.0001.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Comparison

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Venn diagram showing overlap in the identity of proteins with significantly altered abundance in lysosomes isolated from GBA1 -p.E326K KI, GBA1 -p.L444P KI HEK293T, and GBA1 KO cells, relative to lysosomes isolated from WT cells. B Volcano plots showing differential abundance of proteins identified in Lyso-IP samples from indicated GBA1 variant or KO cells compared to WT cells. Proteins with significantly changed abundance (p < 0.05; absolute log 2 FC ≥ 0.5) are highlighted in red or blue for increased or decreased abundance, respectively. Proteins identified as mitochondrial in origin in the MitoCarta 3.0 database are highlighted in purple. C The number of proteins identified in each GBA1 variant or KO genotype belonging to the top ten most significant GO: Cell Component terms associated with the subset of significantly depleted proteins in E326K (vs WT) lysosomes. D Oxygen consumption rate (OCR) of HEK293T cells of indicated genotype, measured using Seahorse XF Cell Mito Stress Test assay. n = 3 independent experiments. E HEK293T cells of indicated genotype were stained with TMRM and MitoTracker Green, and the ratio of the sum (per nuclei) TMRM fluorescence to MitoTracker Green fluorescence, normalized to the mean of all WT cells, is plotted. n = 3 independent experiments. F Heatmap of the relative abundance of all identified glycosphingolipid hydrolases (related to the pathway shown in 2c) is shown as log 2 fold change (from WT mean) for both whole-cell and Lyso-IP samples from HEK293T cells of indicated genotype. Analytes that were not detected in a sample are shaded gray. G Representative immunoblot of Lyso-IP samples from HEK293T cells (of indicated genotype) expressing TMEM192-HA x3 tag. Immunoblots were probed with antibodies against GLA, GM2A, SGSH, and HA as a loading and lysosomal isolation control. H Quantification of lysosomal GLA levels (normalized per sample to HA band intensity), measured via immunoblotting of Lyso-IP fraction & normalized to mean of all WT samples. n = 3 independent experiments. Bar graphs in E and H show mean ± SEM with individual points representative of data from separate experimental replicates. All proteomics data (A-C, F) is from n = 3 independent experiments. Unless otherwise noted, all statistical tests were performed on genotype-level comparisons using robust linear model as described in methods and significance threshold was set at FDR adjusted P value < 0.05. Statistics in E and H were performed on genotype-level comparisons using one-way ANOVA with Dunnett’s multiple comparison test, where * = p < 0.0332, ** = p < 0.0021, **** = p < 0.0001.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Isolation, Variant Assay, Staining, Fluorescence, Western Blot, Expressing, Control, Comparison

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Lysosomal GCase activity, measured in WT, GBA1 -p.E326K, and GBA1 KO human iMicroglia stained with LysoFQ-GBA, is quantified as sum LysoFQ-GBA spot intensity per nuclei, normalized to the mean of all WT cells. n = 3 independent experiments. B Representative immunoblot showing GCase levels in human iMicroglia of indicated genotype. C Quantification of total cellular GCase enzyme levels (normalized to actin) in human iMicroglia of indicated genotype, measured via immunoblot & normalized to mean of all WT cells. n = 3 independent experiments. D Heatmap of whole-cell log 2 fold change (from WT mean) abundance of all detected GCase substrates from human iMicroglia of indicated genotype. Shaded cells indicate values capped at log 2 fold change = 5. E Fold change (over WT mean) of glucosylsphingosine levels in whole-cell samples from human iMicroglia of the indicated genotype. F Heatmap of log 2 fold change (from WT mean) of BMPs & related species from human iMicroglia of indicated genotype. G Heatmap of log 2 fold change (from WT mean) of glycosphingolipids from human iMicroglia of indicated genotype. For F and G, only species with a fold change of E326K vs WT that reached nominal significance (p < 0.1) are included. Lipidomics analysis (D-G) was done using a robust linear model as described in methods and significance threshold was set at FDR adjusted p value < 0.05, from n = 3 independent experiments. Statistics in A, C, and E, were performed on genotype-level comparisons using one-way ANOVA with Dunnett’s multiple comparison test, where * = p < 0.0332, **** = p < 0.0001.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Activity Assay, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: A Common PD-Risk GBA1 Variant Disrupts LIMP2 Interaction, Impairs Glucocerebrosidase Function, and Drives Lysosomal and Mitochondrial Dysfunction

doi: 10.1101/2025.08.28.672891

Figure Lengend Snippet: A Whole blood-derived GCase activity (µmol/L/hr) is plotted in GBA1 -p.E326K carriers (n = 19), GBA1 pathogenic variant carriers (n = 14), and subjects who do not carry either (n = 346). B and C The levels of selected plasma metabolites (adjusted area ratio), glucosylsphingosine ( B ) and GB3 (d18:1/24:0) ( C ), plotted from GBA1 -p.E326K carriers (n = 14), GBA1 pathogenic variant carriers (n = 159), and subjects who do not carry either (n = 410). P-values are derived from linear models comparing GBA1 -p.E326K carriers or GBA1 pathogenic variant carriers to non-carriers, adjusted for age, sex, PD case status, and 3 principal components derived from genome-wide WGS data (see Methods). Gray circles denote controls and colored circles (blue, orange, or red, for non-carriers, p.E326K carriers, and pathogenic variant carriers, respectively) denote PD cases.

Article Snippet: Gba E326K mice (and age-matched WT control mice) were obtained from the Jackson Laboratory (strain #036159).

Techniques: Derivative Assay, Activity Assay, Variant Assay, Clinical Proteomics, Genome Wide

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: Brain

Article Title: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

doi: 10.1093/brain/awu020

Figure Lengend Snippet: Characteristics of patient-derived fibroblasts

Article Snippet: This fits with the hypothesis that E326K reduces glucosylceramidase activity sufficiently to induce alpha-synuclein, but not glucosylceramide, accumulation, thus explaining its association with Parkinson’s disease but not Gaucher disease ( Duran et al. , 2013 ).

Techniques: Mutagenesis, Control

Journal: Brain

Article Title: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

doi: 10.1093/brain/awu020

Figure Lengend Snippet: Glucosylceramidase protein, activity and transcript levels. ( A ) Representative western blots of glucosylceramidase protein. Note reduction in glucosylceramidase protein levels in Gaucher disease (GD) fibroblasts with a lesser degree of reduction in Parkinson’s disease with glucocerebrosidase mutations (PD-GBA), non-manifesting carrier (NMC) and the E326K/E326K fibroblasts. ( B ) Bar chart summarizing glucosylceramidase protein levels as assessed by western blot normalized to control. Bars represent median value and 95% confidence interval for all cell lines in each group; each line was measured on three separate blots. There are significant reductions in Gaucher disease (Mann-Whitney U-test P < 0.001), Parkinson’s disease with glucocerebrosidase mutations ( P < 0.001), non-manifesting carrier ( P = 0.004) and E326K/E326K lines ( P = 0.003). ( C ) Bar chart summarizing glucosylceramidase activity levels (nmol/h/mg). Enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease ( t -test P = 0.001), patients with Parkinson’s disease with glucocerebrosidase mutations ( P = 0.004), non-manifesting carriers ( P = 0.009) and E326k/E326K lines ( P = 0.001) compared with controls. Each bar represents all cell lines in each group. Results are mean ± 1 SD of three separate experiments done for each line. ( D ) GBA transcript levels in disease lines compared to controls ( n = 3), there were reduced transcript levels in GD01, GD04, GD05, and PD03. * P < 0.05.

Article Snippet: This fits with the hypothesis that E326K reduces glucosylceramidase activity sufficiently to induce alpha-synuclein, but not glucosylceramide, accumulation, thus explaining its association with Parkinson’s disease but not Gaucher disease ( Duran et al. , 2013 ).

Techniques: Activity Assay, Western Blot, Control, MANN-WHITNEY

Journal: Brain

Article Title: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

doi: 10.1093/brain/awu020

Figure Lengend Snippet: Evidence of endoplasmic reticulum retention of glucosylceramidase in Gaucher disease. ( A ) Top : immunofluorescence of control cells showing vesicular staining of glucosylceramidase (green) at the periphery of the cell and perinuclear, reticular staining of calnexin (red, endoplasmic reticulum marker). Bottom : co-localization of glucosylceramidase and calnexin represented by yellow perinuclear staining with minimal green vesicular staining pattern in GD02 and GD03 cell lines. ( B ) Representative western blots of cell lysate treated with endoglycosidase-H for 12 h. Note appearance of low molecular weight band in Gaucher disease samples (arrow), which represents endoplasmic reticulum retained glucosylceramidase protein. ‘+’ lanes were treated with endoglycosidase-H. ( C ) Bar chart summarizing percentage of endoglycosidase-H sensitive glucosylceramidase in Gaucher disease (GD) ( t -test P = 0.0001), Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) ( P = 0.01), non-manifesting carriers (NMC) and E326K/E326K ( P = 0.001) compared with controls. Results are mean of three experiments. ( D ) Top : representative western blot of BiP expression. Bottom : representative western blot of calnexin expression. There was significant elevation of endoplasmic reticulum stress markers BiP and calnexin expression in all Gaucher disease, Parkinson’s disease with GBA mutation and non-manifesting carrier lines.

Article Snippet: This fits with the hypothesis that E326K reduces glucosylceramidase activity sufficiently to induce alpha-synuclein, but not glucosylceramide, accumulation, thus explaining its association with Parkinson’s disease but not Gaucher disease ( Duran et al. , 2013 ).

Techniques: Immunofluorescence, Control, Staining, Marker, Western Blot, Molecular Weight, Expressing, Mutagenesis

Journal: Brain

Article Title: Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

doi: 10.1093/brain/awu020

Figure Lengend Snippet: Oxidative stress assays in Gaucher and Parkinson’s disease fibroblasts. ( A ) Graph summarizing increases in rates of dihydroethidium oxidation rates for Gaucher disease (GD) (Mann-Whitney U-test P < 0.001), Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) ( P < 0.001), non-manifesting carriers (NMC) ( P < 0.001) and E326K/E326K ( P < 0.001) compared with control cells. ( B ) Graph demonstrating significant reduction in dihydroethidium oxidation rates for controls ( n = 2), Gaucher disease ( n = 3) and Parkinson’s disease with glucocerebrosidase mutations fibroblasts ( n = 2) treated with ambroxol compared to untreated cells. Results expressed as median rate of dihydroethidium oxidation ± 95% confidence interval.

Article Snippet: This fits with the hypothesis that E326K reduces glucosylceramidase activity sufficiently to induce alpha-synuclein, but not glucosylceramide, accumulation, thus explaining its association with Parkinson’s disease but not Gaucher disease ( Duran et al. , 2013 ).

Techniques: MANN-WHITNEY, Control