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(A) Diagram of iPSC cell line generation and differentiation protocol into midbrain dopaminergic neurons and astrocytes, with r epresentative images of immunocytochemistry of GBA1 +/+ iPSC-neurons stained with anti-TH (green) and DAPI and GBA1 +/+ iPSC-astrocytes stained <t>with</t> <t>anti-S100β</t> (red) and DAPI (B) Quantification of glucocerebrosidase (GCase) enzyme activity by relative fluorescence of 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) in indicated genotypes. GCase activity of all cell lines treated with CBE was subtracted to eliminate background activity. (one-way ANOVA F (4, 19) =9.092, p=0.0003). (C) Representative Western blot with anti-GBA1 protein in GBA1 +/+ , GBA1 IVS/+ , GBA1 +/+ + conduritol B epoxide (CBE), and GBA1 IVS/IVS iPSC-neurons. (D) Quantification of GBA1 protein in neurons from 3 independent replicates in indicated genotypes, normalized to Actin in control (one-way ANOVA F (3,8) =108.6, p<0.0001). (E) Heatmap of differentially expressed midbrain dopaminergic neuron-and astrocyte-specific genes in GBA1 +/+ neurons versus GBA1 +/+ astrocytes sorted by z-score. Statistical significance determined by one-way ANOVA (ns = p ≥ 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Data are presented as mean ± SEM.
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(A) Diagram of iPSC cell line generation and differentiation protocol into midbrain dopaminergic neurons and astrocytes, with r epresentative images of immunocytochemistry of GBA1 +/+ iPSC-neurons stained with anti-TH (green) and DAPI and GBA1 +/+ iPSC-astrocytes stained <t>with</t> <t>anti-S100β</t> (red) and DAPI (B) Quantification of glucocerebrosidase (GCase) enzyme activity by relative fluorescence of 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) in indicated genotypes. GCase activity of all cell lines treated with CBE was subtracted to eliminate background activity. (one-way ANOVA F (4, 19) =9.092, p=0.0003). (C) Representative Western blot with anti-GBA1 protein in GBA1 +/+ , GBA1 IVS/+ , GBA1 +/+ + conduritol B epoxide (CBE), and GBA1 IVS/IVS iPSC-neurons. (D) Quantification of GBA1 protein in neurons from 3 independent replicates in indicated genotypes, normalized to Actin in control (one-way ANOVA F (3,8) =108.6, p<0.0001). (E) Heatmap of differentially expressed midbrain dopaminergic neuron-and astrocyte-specific genes in GBA1 +/+ neurons versus GBA1 +/+ astrocytes sorted by z-score. Statistical significance determined by one-way ANOVA (ns = p ≥ 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Data are presented as mean ± SEM.
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(A) Diagram of iPSC cell line generation and differentiation protocol into midbrain dopaminergic neurons and astrocytes, with r epresentative images of immunocytochemistry of GBA1 +/+ iPSC-neurons stained with anti-TH (green) and DAPI and GBA1 +/+ iPSC-astrocytes stained with anti-S100β (red) and DAPI (B) Quantification of glucocerebrosidase (GCase) enzyme activity by relative fluorescence of 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) in indicated genotypes. GCase activity of all cell lines treated with CBE was subtracted to eliminate background activity. (one-way ANOVA F (4, 19) =9.092, p=0.0003). (C) Representative Western blot with anti-GBA1 protein in GBA1 +/+ , GBA1 IVS/+ , GBA1 +/+ + conduritol B epoxide (CBE), and GBA1 IVS/IVS iPSC-neurons. (D) Quantification of GBA1 protein in neurons from 3 independent replicates in indicated genotypes, normalized to Actin in control (one-way ANOVA F (3,8) =108.6, p<0.0001). (E) Heatmap of differentially expressed midbrain dopaminergic neuron-and astrocyte-specific genes in GBA1 +/+ neurons versus GBA1 +/+ astrocytes sorted by z-score. Statistical significance determined by one-way ANOVA (ns = p ≥ 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: GBA1 deficiency differentially affects endolysosomal trafficking in neurons versus astrocytes

doi: 10.64898/2026.01.12.697563

Figure Lengend Snippet: (A) Diagram of iPSC cell line generation and differentiation protocol into midbrain dopaminergic neurons and astrocytes, with r epresentative images of immunocytochemistry of GBA1 +/+ iPSC-neurons stained with anti-TH (green) and DAPI and GBA1 +/+ iPSC-astrocytes stained with anti-S100β (red) and DAPI (B) Quantification of glucocerebrosidase (GCase) enzyme activity by relative fluorescence of 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) in indicated genotypes. GCase activity of all cell lines treated with CBE was subtracted to eliminate background activity. (one-way ANOVA F (4, 19) =9.092, p=0.0003). (C) Representative Western blot with anti-GBA1 protein in GBA1 +/+ , GBA1 IVS/+ , GBA1 +/+ + conduritol B epoxide (CBE), and GBA1 IVS/IVS iPSC-neurons. (D) Quantification of GBA1 protein in neurons from 3 independent replicates in indicated genotypes, normalized to Actin in control (one-way ANOVA F (3,8) =108.6, p<0.0001). (E) Heatmap of differentially expressed midbrain dopaminergic neuron-and astrocyte-specific genes in GBA1 +/+ neurons versus GBA1 +/+ astrocytes sorted by z-score. Statistical significance determined by one-way ANOVA (ns = p ≥ 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Data are presented as mean ± SEM.

Article Snippet: After 2 weeks in Astrocyte Maturation Medium, a small portion of cells was fixed and stained to evaluate the presence of mature astrocyte markers: mouse anti-glial fibrillary acidic protein (GFAP) (Sigma-Aldrich G3893, 1:400), mouse anti-Vimentin (Invitrogen #MA5-11883, 1:500), rabbit anti-Vimentin (Cell Signaling Technology 5741S, 1:100), rabbit anti-S100β (Proteintech 15146-1-AP, 1:100), and rabbit anti-Aquaporin 4 (AQP4Invitrogen #PA5-85767, 1:100).

Techniques: Immunocytochemistry, Staining, Activity Assay, Fluorescence, Western Blot, Control