Fig. 1.

SLC35B2 is required for endocytosis of α-Syn PFF. (A) The CRISPR screen strategy. (B–D) SLC35B2-KO cells are defective in endocytosis of α-Syn PFF but not monomeric α-Syn. (B) Validation of SLC35B2 (35B2)-KO cells by qRT-PCR. mRNA levels were normalized to the level in control cells. The error bar represents SEM. n = 3. (C) Control, SLC35B2-KO, or SLC35B2-KO cells stably expressing FLAG-SLC35B2 were incubated with pHrodo-labeled α-Syn PFF (200 nM for 4 h) and analyzed by FACS. FL, fluorescence. (D) Control and SLC35B2-KO cells were treated with labeled α-Syn monomer at 800 nM overnight before FACS analysis. (E–G) Knockdown (KD) of SLC35B2 attenuates α-Syn PFF uptake in primary neurons. (E) Primary neurons were infected with lentivirus expressing the indicated shRNAs together with EGFP (driven by the synapsin promoter) at day in vitro (DIV) 3. mRNA was purified from cells at DIV8 for qRT-PCR analysis. Error bar represents SEM. n = 2 biological repeats, each with triplicate PCR analyses. (F) Control or SLC35B2-KD neurons expressing EGFP (green) at DIV8 were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) (red) for 4 h, stained with DAPI (blue), and analyzed by confocal microscopy (Scale bar: 5 µm.) (G) Quantification of α-Syn PFF level in individual cells (indicated by dots) from two independent experiments. Error bar represents SEM. P value from a two-tailed t test. A.U., arbitrary unit. (H and I) SLC35B2 is required for α-Syn PFF binding to the plasma membrane. (H) Control, SLC35B2-KO, or SLC35B2-KO cells reexpressing WT SLC35B2 were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) (magenta) on ice for 30 min, stained with DAPI (blue), and imaged by confocal microscopy (Scale bar: 5 µm.) (I) Quantification of α-Syn PFF surface level in individual cells from two independent experiments.