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. 2021 Nov 24;7(48):eabj1826. doi: 10.1126/sciadv.abj1826

Fig. 2. PrP-directed antibodies cause increased ADAM10-mediated PrP shedding in N2a cells.

Fig. 2.

(A) PrP scheme showing important domains (CC, charged cluster; OR, octameric repeat region; HD, hydrophobic domain), GPI anchor position, shedding, and α-cleavage sites, plus epitopes for antibodies used here. aa, amino acids. (B) Prnp mRNA levels in cells either untreated or treated for 16 hours with indicated antibodies or single-chain (sc) derivates. Negative controls: PrP-depleted cells (Prnp KO). n = 3 independent experiments (n = 2 for Prnp KO) with three technical replicas each. No significant differences in Prnp mRNA levels were found among different treatments and untreated controls. (C) Representative immunoblot analysis of fl-PrP in lysates (bottom) and sPrP in precipitated medium (top) after 16 hours of incubation with different PrP-directed IgGs. Loading controls: β-actin (lysates) and sAPPα (medium). fl-PrP levels were reduced (P ≤ 0.05) only in POM2-treated cells compared to secondary antibody controls, whereas significantly increased sPrP/fl-PrP ratios were observed for 6D11 and POM1 treatment (P ≤ 0.0001). Data show means ± SEM of n = 5 independent experiments; statistical significance was estimated with analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. (D) Microscopy of untreated and treated cells showing no alterations in density or overall morphology (scale bar, 100 μm). (E) Treatment with POM2 or POM1 in the presence (+GI254023X) or absence [+DMSO (dimethyl sulfoxide), as diluent control] of an ADAM10 inhibitor (left). Right: N2a WT or N2a stably expressing murine PrP with the human 3F4 epitope (N2a PrP3F4) treated or not with 3F4 antibody targeting that motif. Shedding only increased in PrP3F4-expressing cells. (F) Ascending concentrations of 6D11 reveal a dose dependency of the shedding-stimulating effect (reaching saturation at ~1 μg/ml). (G) The bispecific immunotweezer (scPOM-bi; fused complementarity-determining regions VH/VL of POM2 and POM1; see scheme) increases shedding compared to untreated controls. Quantification with controls set to 1 (mean ± SE; *P = 0.024, Student’s t test). Positive control: 6D11 treatment [reduced levels of sPrP-C1 fragment (asterisk) possibly due to 6D11 sterically hindering α-cleavage before shedding]. *P < 0.05 and ****P < 0.0001.