Fig 2: Investigation of the binding properties of single-cell gC1q (sc-gC1q) variants by ELISA. Human and mouse IgG proteins (hIgG and mIgG, respectively) and human IgM were immobilized and titrated with each form of sc-gC1q variants (A, B, and E). In order to use the same primary antibody in assays examining neuronal pentraxins, we coated the wells with NPTX1 or NPTX2 and titrated with the different forms of sc-gC1q (C and D; marked with different colors). Hill equations (lines) were fitted to the data (circles) using Origin8 software. Values represent mean (from three replicates) ± SEM. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin.

Fig 3: SPR-binding studies. After covalent immobilization of IgG, IgM, NPTX1, and NPTX2) via amine coupling, the ligand channels were covalently blocked with bovine serum albumin (BSA) along with an empty channel that served as negative control. The sensorgrams (pale lines) are generated with the subtraction of the responses detected on the BSA channel from those detected on specific ligand channels. Dark lines represent the data fitting with a single exponential (sc-gC1q2 and sc-gC1q2l) or a double exponential (sc-gC1q). A–D, sc-gC1q analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. E–H, sc-gC1q2 analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. I–L, sc-gC1q2l analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin; sc-gC1q, single-chain gC1q; SPR, surface plasmon resonance.

Fig 4: Co-occurrence of the C1q-tag with NP1/2 on synaptosomes examined by flow cytometry. According to the gating criteria, C1q-labeled synaptosomes were almost exclusively (~97%) positive for NP1 (A) and NP2 (B). In contrast to C1q labeling, only ~24% of NP-labeled synaptosomes were positive for C1q. Density plots show representative measurements where the percentages of synaptosomes that belong to each quadrant were also indicated. The secondary antibody controls went through the same procedure as the fully labeled samples. Statistically significant differences were determined with two-tailed Student’s t-test of paired samples (Means ± SEM; n = 4 mice). To test the viability of synaptosomes, calcein-AM labeling was applied separately ( Supplementary Datasheet 2 ).

Fig 5: Longitudinal changes from baseline in cerebrospinal fluid concentration of neuronal pentraxin 2 (NPTX2) (raw data from individual participants: thin lines; mean changes resulting from the linear mixed effects (LME) model: thick lines, also shown in Figure 1 under “NPTX2”). A, Participants categorized as cognitively normal (CN) at baseline (blue); participants categorized as mild cognitive impairment (MCI) at baseline (red). B, Participants categorized as phosphorylated tau (p‐tau)181/amyloid beta (Aβ)1‐42 ratio positive at baseline (red); participants categorized as p‐tau181/Aβ1‐42 ratio negative at baseline (blue). C, Participants categorized as progressors (red); participants categorized as non‐progressors (blue)