Project 2: How does human C4 allelic diversity affect microglia-mediated synaptic pruning? Abstract: Schizophrenia (SCZ) patients exhibit excessive loss of grey matter and decreased dendritic spine density, suggesting that abnormal synaptic pruning may contribute to SCZ etiology. Genetic studies indicate that complement C4A is a susceptibility factor for SCZ, although it is not clear if this link is a result of increased complement expression in the brain or in the periphery, or both. The complement system is a major mediator of inflammation in the periphery and over expression of C4 could induce release of pro-inflammatory cytokines that induce pathology affecting both the periphery and the CNS. The human C4 locus encodes two highly conserved isoforms named C4A (acidic) and C4B (basic) which differ overall by only four amino acid differences. The differences are functionally important in binding to target sites. In the brain, C4 localizes to synapses and is required for synaptic pruning in the developing visual system. Whether the two isoforms differ in binding efficiency to synapses has not been reported. SCZ patients express elevated levels of C4A relative to C4B in the CNS suggesting that the level of local expression of C4 in the brain may also be an important factor in disease. To help address these questions, we have developed novel strains of mice that express the common alleles of human C4, i.e. C4A only, C4B only or C4A and C4B, and crossed them to the C4-deficident background. In addition, we have used gene editing to alter the murine C4 locus to express the human C4A- or C4B-like isotypic region. Given the importance of complement C4 in synapse elimination in the murine visual system, will use these mice to test our hypothesis that chemical differences in C4 combined with increased level of local expression affect binding to synapses and directly affect the efficiency of synapse elimination by activated microglia. Two specific aims are proposed: Aim 1: How do the activities of C4A and C4B differ in the periphery and CNS? This aim will precisely define how C4A and C4B functional differences affect developmental synaptic pruning and microglia activity in the LGN. Aim 2: What are the effects of complement overexpression on synaptic pruning? This aim will test for the first time the idea that complement overactivation stimulates excessive synaptic pruning using mouse models designed to model C4A expression in SCZ.