Fig. 4
From: Single-domain antibody-based protein degrader for synucleinopathies
Enhanced intracellular clearance of α-syn by modified sdAb 2D8-PEG4-T in primary neurons. (A) M83 primary neurons were incubated with 10 µg/ml LBD S1 fraction for 24 h. Subsequently, different concentrations of sdAbs ranging from 0.625–10 µg/ml of 2D8 and molar equivalent of modified 2D8 were added, with samples collected 96 h post-antibody application. (B) Schematic diagram of PROTAC-mediated ternary complex formation and Hook effect based on PROTAC concentration. (C) One-way ANOVA analysis revealed significant differences in normalized total α-syn levels when sdAb concentration exceeded 0.625 µg/ml (p = 0.02 for 1.25 µg/ml, p = 0.0004 for 2.5 µg/ml, p = 0.0001 for 5.0 µg/ml, p = 0.0136 for 10.0 µg/ml). At 1.25–5.0 µg/ml sdAb concentration, S1 alone increased α-syn levels by 46% compared to control at 96 h (p = 0.0154 (1.25 µg/ml), p = 0.0049 (2.5 µg/ml), p = 0.0043 (5.0 µg/ml)). At 2.5–5.0 µg/ml, 2D8-PEG4-T reduced S1-induced increase in total α-syn levels (reduced by 61% at 2.5 µg/ml, p = 0.0001, and 67% at 5.0 µg/ml, p < 0.0001, relative to S1 alone). Modified sdAb 2D8-PEG4-T also lowered total α-syn levels compared to unmodified 2D8 at 2.5 and 5.0 µg/ml (reduced by 38% at 2.5 µg/ml, p = 0.0298, and 41% at 5.0 µg/ml, p = 0.0126, relative to unmodified 2D8). (D) Immunoblots depicting total α-syn and GAPDH levels in treated M83 cell lysate. Complete western blots and bands analyzed are in Supplemental Fig. 6. (E) Immunofluorescence of M83 primary mouse neurons treated with 10 µg/ml LBD S1 fraction for 24 h. Media was then exchanged, and unmodified sdAb 2D8 or modified sdAb 2D8-PEG4-T at 5.0 µg/ml or molar equivalent were added and incubated for an additional 2 h. Neurons were subsequently immuno-probed for sdAb containing His tag (HIS.H8, green) and total α-syn (211, red). Scale bar: 20 μm. Mander’s coefficient M2 quantifies the ratio of α-syn overlapping with sdAb. Calculated as the sum of overlapping intensity in α-syn divided by total intensity of α-syn. *, **, ***, ****: p < 0.05, 0.01, 0.001, 0.0001 (Tukey’s post-hoc test)