Fig 1: The 2A7 antibody detects aSyn expression in neural precursors and neurons. (A) The 2A7 antibody detects higher aSyn protein levels in neurons (TUBB3+) compared to non-neuronal cells (TUBB3-) in hiPSC-derived cortical organoids. Left: Histograms of intracellular aSyn staining of hiPSC-derived cortical organoid cells. Right: Comparison of mean fluorescence intensities (MFI) of intracellular aSyn staining in TUBB3+ and TUBB3- organoid cells. Error bars represent standard deviations. Four independent staining experiments were performed. (B) Intracellular aSyn staining with the 2A7 antibody in the midbrain and cortical neural precursor cells (NPCs) revealed a more robust detection of an aSyn protein increase in NPC from patients with Parkinson's disease having SNCA locus duplication (Dupl 1, 2, and 3 lines) compared to control NPC (Ctrl 1 and 2 lines). Histograms (“midbrain NPCs” and “cortical NPCs”) and quantified mean fluorescence intensities (MFI, right to each histogram) are shown. Normalized MFI values for each staining were calculated as a difference between MFI of antibody staining and of isotype control staining (Iso). (C) The 2A7 antibody detects a significant increase of aSyn expression in hiPSC-derived midbrain dopaminergic neurons from SNCA locus duplication in patients with Parkinson's disease (SNCA dupl) compared to control (ctrl) using immunocytochemistry. Left: Representative images of ctrl (Ctrl 1 line) and SNCA dupl hiPSC-derived (Dupl 1 line) midbrain dopaminergic neurons stained for aSyn (green) with 2A7 antibody, tubulin ß3 (Tuj 1; white), and DAPI (blue). Right: Quantification of mean gray values of the 2A7 aSyn staining of ctrl and SNCA dupl midbrain dopaminergic neurons. Error bars represent standard deviations. Five independent measurements were performed. **p < 0.01, one-way ANOVA.
Fig 2: SNCA accumulation is resistant to enzyme replacement and substrate reduction therapy.(A) Mean cathepsin D activity in WT cells and 2 KO clones showing no differences in enzyme activity (n = 3). (B) Western blots of SNCA and TUBA in vehicle- and aGAL-treated WT and KO cells with quantification confirming the overexpression of SNCA protein and its resistance to aGAL treatment (n = 4). (C) Human SNCA ELISA depicting SNCA accumulation in KO clones and resistance to substrate reduction therapy using Venglustat (n = 5). (D) SNCA staining in representative images and quantification of human renal biopsies showing increase in untreated Fabry samples with resistant accumulation in patients who underwent 5 years of ERT (n = 5). Scale bars: 50 µm. **P < 0.01, ****P < 0.0001. One-way ANOVA with Tukey’s multiple-comparison test.
Fig 3: SNCA mediates observed lysosomal dysfunction.(A) Representative Western blot confirming the efficacy of siRNA targeting SNCA in WT and KO clones (n = 4). (B) Quantification of lysosomal area, pH, and ROS production upon SNCA siRNA treatment (n = 18). (C) Representative Western blot confirming the overexpression of SNCA in WT cells (n = 4). (D) LAMP1 immunofluorescence staining shows an increase in lysosomal aggregation upon SNCA overexpression (arrowhead). Scale bars: 10 µm. (E) Quantification of lysosomal area (n = 20), pH (n = 8), and ROS production (n = 12) upon SNCA overexpression. Violin plots indicate median (red) and upper and lower quartile (blue). *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001. One-way ANOVA with Tukey’s multiple-comparison test (B); unpaired, 2-tailed Student’s t test (E).
Fig 4: ß2-Adrenergic receptor agonists decrease SNCA protein levels and ameliorate lysosomal dysfunction.(A) Connectivity mapping showing anti-Fabry compounds, with the ß2-adrenergic receptor agonist orciprenaline exhibiting the highest score. (B) Western blots show the expression of SNCA in WT and untreated GLA-KO cells and KO cells treated with 20 µM clenbuterol and 10 µM orciprenaline (n = 6). (C) Western blots depict the expression of LAMP1 and ACTN4 in WT and untreated GLA-KO cells and KO cells treated with aGAL, 20 µM clenbuterol, and combined therapy (n = 6). (D) Lysosomal pH analysis in all conditions demonstrates independent and additive effects of ß2-adrenergic receptor agonist in GLA-KO cells (n = 6). (E) Lysosomal ROS analysis demonstrates independent and additive effects of ß2-adrenergic receptor agonist in GLA-KO cells (n = 6). (F) Schematic summary depicting the overall findings of the study: Fabry podocyte lysosomes are characterized by increased size, pH, and ROS production with subsequently decreased function due to Gb3 and SNCA accumulation. This phenotype can be ameliorated through ERT combined with compounds decreasing SNCA accumulation, like ß-receptor agonists. Bar graphs depict standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey’s multiple-comparison test.
Fig 5: SILAC-based proteomics and network analyses identify SNCA accumulation as a potential pathogenic pathway.(A) Schematic overview of mass spectrometry analysis using SILAC-labeled WT and KO clones. Mass spectrometry yielded 2,248 proteins, among which 321 are lysosome-enriched. (B) The top 10 up- and downregulated lysosome-enriched proteins. (C) Network-based analysis of up- and downregulated lysosomal proteins associated with GLA knockout. Nodes represent genes and are connected if there is a known protein interaction between them. The node size is proportional to the number of its connections. Red and blue nodes represent up- and downregulated seed proteins, respectively. Light red and light blue nodes represent the respective DIAMOnD proteins. GLA is depicted as a green node. Pink nodes indicate shared proteins between the 2 modules. The separate network and Venn diagram on the right show the number and interaction partners of SNCA.
Supplier Page from Abcam for Human Alpha-synuclein ELISA Kit