Increased activation product of complement 4 protein in plasma of individuals with schizophrenia

Abstract

Structural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood-brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

Conflict of interest statement

Dr. Jacob Hall receives speaker fees from Biogen. The remaining authors declare no competing interests.

© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.

Figures

Fig. 1. C4A/B genes produce C4A/B protein, which both release C4-ana when activated.

A C4-ana is produced when C4 protein is activated in the Classical and Lectin Complement Pathways. Similarly, C3-ana is released when C3-convertase is activated and C5-ana is produced upon activation of the Terminal Complement Pathway. B An inactive retrovirus, HERV, present between exons 9 and 10 of the C4A or C4B gene, differentiates the long form of the gene from the short form of the gene. The C4A gene produces the C4A (acidic) protein while the C4B gene produces the C4B (basic) protein, differentiated by 5 amino acids as a result of four nucleotide polymorphisms. Both forms of the protein lead to the cleavage product, C4-ana when activated by binding to protein binding partners.

Fig. 2. C4-ana concentration is higher in plasma from individuals with schizophrenia.

A Concentration of C4-ana, C3-ana, and C5-ana in plasma in the whole cohort. The mean concentration of C4-ana is higher for the patient group compared to controls, p = 0.01 (p = 0.02 if the outlier is removed), as calculated by Mann-Whitney. The mean plasma levels of C3-ana, C5-ana are lower in the control group when compared to the group from individuals with schizophrenia. Detailed values and statistics are shown in the table in B.

Fig. 3. The relationship between C4-ana concentration and C4 gene copy number differs in cases vs. controls.

A The number of C4AL, C4AS, C4BL, and C4BS genes were determined experimentally by ddPCR. The frequency of each gene variant in the control and individuals with schizophrenia groups are reported. Total C4A was determined by adding the number of C4AL and C4AS gene copies for each participant. Similarly, C4B was determined by adding the number of C4BL and C4BS gene copies for each participant. Total C4 was determined by adding the total gene copies of C4A and C4B for each participant. The frequencies of each gene copy number for participants are reported in the graphs above. The average gene copy numbers for each distribution are reported in table (B). In this cohort, we see that samples from individuals with schizophrenia have higher gene copy numbers of total C4A and C4AL genes compared to controls (p-value = 0.04 and 0.03, respectively. These p-values are not statistically significant if the Bonteferoni correction is applied). C The table shows results of Spearman’s rho correlation analyses between plasma C4-ana concentration and (i) each subset of C4 gene copy numbers (total C4, C4A, C4B, C4AL, C4AS, C4BL, C4BS); and (ii) predicted C4A and C4B gene expression in brain based on the formula of Sekar et al. D Plasma C4-ana concentration (Y axis) is plotted against total C4A (X axis), with regression lines shown for the entire cohort and separately for cases and controls. Cases showed a positive relationship, and controls an inverse relationship, between C4A GCN and plasma C4A GCN.

Fig. 4. C4-ana increases the permeability of primary brain endothelial cells.

A Brain endothelial cells stained with phalloidin and DAPI. After BEC are exposed to C4-ana, cells form actin stress fibers. B The permeability of the monolayer increases after exposure to increasing concentrations of C4-ana and thrombin.