Abstract: Multiple lines of evidence suggest that increased synaptic pruning is pathophysiologically significant for schizophrenia (SZ). Emerging evidence suggests that genetic factors are associated with increased synaptic pruning in SZ. Recent genome-wide association studies convincingly demonstrated that Complement (C4) gene repeats were significantly associated with SZ risk, specifically C4A long form (C4AL). C4 protein expression in the brains of schizophrenia patients was increased proportionate to the number of C4AL repeats. A mouse analog of C4 was associated with increased synaptic pruning in a rodent model. We propose to develop two independent lines of evidence on the association of genetic factors with synaptic pruning. Our in vivo approach will longitudinally examine measures of synaptic pruning obtained using ultra-high field structural MRI and phosphorus magnetic resonance spectroscopy (31P MRS) at 7 Tesla at baseline, 6-months and 1-year among adolescents with early onset SZ (EOS) and healthy controls (HC), and its association with cognitive performance (Aim 1), C4 repeats and global genetic liability measured through polygenic risk score (PGRS) (Aim 2). The rationale for longitudinal examination of adolescent EOS and HC is supported by more prominent synaptic pruning during adolescence. Ultra-high field structural MRI and 31P MRS at 7 Tesla provides superior resolution and greater sensitivity for metabolite quantification. 31P MRS measures metabolites of membrane phospholipids (MPL) across the entire brain. Decreased MPL precursors index decreased neuropil formation whereas increased MPL breakdown products reflect neuropil contraction. Since synapses are embedded in the neuropil and major part of membrane expansion is contributed by synapses, dendritic branches and dendritic spines, MPL metabolite levels are proposed to provide more specific and sensitive measure of neuropil compared to gray matter metrics. An additional measure to index neuropil contraction is through cortical thickness measurements. PGRS is a more accurate measure of overall genetic risk conferred by genomewide variations. Our in vitro approach will examine dendritic spine density and morphology in relation to expression of C4 proteins - both naturalistically and with induction of over- and under- expression of C4 proteins in differentiating human induced pluripotent stem cell (hiPSC)-derived neuronal organoids (Aim 3). Organoids are 3-dimensional neuronal cultures that show more refined microanatomic feature capable of recapitulating some specific functions of the brain compared to 2-dimensional cultures. In addition to providing independent evidence of synaptic density related to C4 protein expression, hiPSC model provides some corroborative evidence for the 31P MRS and cortical thickness findings that may represent synaptic pruning. Proposing only one line of evidence leaves open questions about the association of genetic factors with neuropil pruning. Examining an important pathophysiological process, namely synaptic pruning, in relation to genetic risk may help identify critical pathways and specific molecular targets for eventual development of novel treatments for SZ.

Public Health Significance Increased synaptic pruning is associated with schizophrenia. Understanding factors associated with pruning may reveal critical pathways for new treatments. Recent genome-wide association study reported the association of variants on Complement 4 (C4) gene with schizophrenia and in animal model with synaptic pruning. We propose to obtain two independent lines of evidence by longitudinally examining the association of measures of synaptic pruning in a human context among adolescent early onset schizophrenia (EOS), and concurrently examine a cellular model using human induced pluripotent stem cell-derived neuronal organoids for synaptic density associated with C4 protein expression. These two independent lines of evidence on genetic factors associated with synaptic pruning may be further investigated to develop novel treatments.