Journal: Translational Neurodegeneration
Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes
doi: 10.1186/s40035-025-00518-0
Figure Lengend Snippet: Expression of CAR-A and effect on α-synO phagocytosis and digestion. a Design of the CAR-expression plasmid. CAR was expressed in fusion with 3A scFv and enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific promotor GfaABC1D. SP, signal peptide; Poly(A), polyadenylation signal; ORI, origin of replication; KanR, kanamycin resistance gene. b Representative image of CAR expression on an astrocyte. The co-localization of 3A, MerTK and EGFP was assessed by confocal microscopy. Scale bars, 10 μm. c Flow cytometry analysis of the binding of CAR-A and ns-CAR-A to α-syn monomers and oligomers (α-synOs). The astrocytes were transfected with CAR or ns-CAR lipoplexes for 48 h. After 2 h-incubation with 1 μmol/L α-syn monomers (α-syn) or α-synOs, cells were stained with PE-labeled anti-α-syn antibody. d PE fluorescence in EGFP-positive astrocytes. n = 3 independent experiments. e Flow cytometry analysis of the amount of α-synO engulfed by CAR-A, ns-CAR-A and NC-A in the presence of different α-synO concentrations. n = 3 independent experiments. f Representative images depicting the phases of engulfment and digestion of α-synO by CAR-A. CAR-A was treated with 1 μmol/L α-synO, and the medium was changed after 1 h incubation. α-SynO and Lamp1 in CAR-A were stained with respective antibodies at different time points and imaged by confocal microscopy. Scale bars, 5 μm. g The kinetic curves of α-synO digestion in CAR-A, ns-CAR-A and NC-A. n = 3 independent experiments. h Statistical analysis of the proportion of α-syn colocalized with Lamp1 in digestion stage in ( f ) by Image J. n = 4 independent experiments. i Intracellular α-syn in Triton X-100-soluble and -insoluble fraction detected by Western blotting at different time points post astrocytic phagocytose of α-synOs. β-actin was used as a control. j Quantification of α-syn ( i ) using Image J. n = 3 independent experiments. k Representative images depicting the binding of ns-CAR-A, NC-A and CAR-A to α-syn monomers and oligomers. Scale bars, 5 μm. Data are mean ± S.E.M. One-way ANOVA ( d ) or Two-way ANOVA ( e ) followed by Tukey’s multiple comparison test was used for statistical analysis. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicate significance compared to respective groups
Article Snippet: In the first construct, the enhanced CMV promotor sequence in the MerTK expression plasmid (purchased from Sino Biological Inc., #MG50514-ACG) was replaced with GfaABC1D promotor sequence synthesized from Sangon.
Techniques: Expressing, Plasmid Preparation, Control, Confocal Microscopy, Flow Cytometry, Binding Assay, Transfection, Incubation, Staining, Labeling, Fluorescence, Western Blot, Comparison
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