Fig 2: Prolonged anesthesia caused synaptic elimination in the hippocampus by complement-mediated synaptic elimination and decreased dendritic spines and their genes. A Prolonged anesthesia increased complement-mediated synaptic elimination in the hippocampal CA1 region. Complement C1qa (green) colocalized with PSD95 (red) puncta was elevated after prolonged anesthesia. Scale bar = 10 μm. B Prolonged anesthesia reduced synaptic density in the hippocampal CA1 region. SYN1 (red), as a marker of synaptic density, was sharply decreased after prolonged anesthesia. Scale bar = 10 μm. C SEM (scanning electron microscope) identifies ultrastructural alteration of synapses in the hippocampal. Synaptic vesicles were reduced, and postsynaptic membrane continuity was interrupted after prolonged anesthesia. Scale bar = 500 nm. D The number of C1qa-colocalized PSD95 between both groups (n = 4 per group). E Prolonged anesthesia reduced the number of synapses. The synapse was marked by presynaptic (SYP) and postsynaptic protein (PSD95). Synapse partially enlarged as shown in panel E. Scale bar = 10 μm. F Synaptic density in the hippocampal CA1 region between both groups (n = 5 per group). G The number of synapses in the hippocampal CA1 region between both groups (n = 4 per group). H, I Prolonged anesthesia downregulated genes of the dendrite (H) and dendritic spines (I). In panels H to I, the horizontal axis is the z-score of the sample, and the vertical axis is the gene. The warmer the color block is, the higher the expression level is, and the colder the color block is, the lower the expression level is (n = 3 per group). J Golgi staining in the hippocampal CA1 region. Scale bar = 100 μm. K Prolonged anesthesia triggered the loss of dendritic spines. L Maximal intersection number per neuron between groups (n = 3 per group). M Sholl analysis discovered that prolonged anesthesia decreased maximal intersection number per neuron between groups. Scale bar = 50 μm. N The number of dendritic spines in the hippocampus between groups (n = 4 per group). Data was shown as Mean ± SD, with *P < 0.05 or *P < 0.001; Sevo group vs. control group. Arrows represent synaptic puncta or colocalization

Fig 3: Competition of sc-gC1qs with full-length C1q binding to neuronal cell surface. Representative confocal fluorescence microscopy images are shown (A–D). After incubation with full-length C1q, cells were treated with sc-C1q-free medium (A), medium containing sc-gC1q (B), sc-gC1q2 (C), and sc-gC1q2l (D). Detection of full-length C1q and separation from sc-gC1q was based on applying primary antibody specific to C1q tail region. E, histograms of fluorescence intensities were determined by ImageJ program. Normalization was performed between solely secondary antibody–treated (sec. crtl) experiment and uninhibited measurement with full-length C1q treatment (flC1q). All three sc-gC1qs lowered significantly the full-length C1q signal in immortalized neural cell culture. One-way ANOVA was performed with post hoc Tukey’s test for mean comparison between flC1q and each sc-gC1q treatments. At the 0.05 level, the population means are different (F = 17). Sc-gC1q, sc-gC1q2, and sc-gC1q2l significantly lowered flC1q signal (p = 3 × 10−7, p = 0.001, and p = 1.16 × 10−5, respectively). Values represent mean ± SEM (from 11 images from uninhibited and inhibited samples and two images from secondary control; two individual experiments were performed from which five and six images were taken). gC1q, globular part of C1q; sc-gC1q, single-cell gC1q.

Fig 4: 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 5: Single-chain gC1q (sc-gC1q) and its multimeric constructs.A, sequence of monomer and multimer sc-gC1q proteins. Color coding is marked below the sequences. Dimer proteins consist of a sc-gC1q and GCN4 leucine zipper as dimerizing module, whereas trimeric constructs contain the trimerizing neck region of surfactant protein D (SpD). Globular head domains of C1q are linked by 4 to 4 amino acid long linkers (GTGG between C1qA and C1qC chains; GSGA: between C1qC and C1qB chains). Both dimer and trimer constructs were designed with shorter (five residues: GSAAA) and longer (13 residues: GSASGSGSGSSAA) linkers fused to sc-gC1q sequence. B, schematic structure of the multimeric constructs. Two or three globular gC1q heads are connected by dimerizing or trimerizing coiled-coil sequences, respectively. C, representative structure from two viewpoints of sc-gC1q: ß-strands of the three domains of jelly-roll fold are shown in blue, red, and yellow. gC1q, globular part of C1q.