14. SINGLE-CELL MULTIOMICS OF NEURON ACTIVATION REVEALS CONVERGENT ACTIVITY-DEPENDENT EFFECTS OF SCHIZOPHRENIA RISK GENES ON LIPID-MITOCHONDRIAL AXIS

Background Genome-wide association studies (GWAS) have identified hundreds of risk loci for schizophrenia (SCZ) and other neuropsychiatric disorders (NPD), yet most disease causal variants and genes remain unknown. A major challenge is that most GWAS risk variants are in noncoding regions and likely act in specific biological contexts. A biological context particularly relevant to NPD is neuronal activation, which has been difficult to study at population scale in human neurons. Methods We modeled neuron activation by using membrane-depolarizing potassium chloride (KCl) to stimulate the co-cultured excitatory (Glut+) and inhibitory (GABA+) neurons of 100 human induced pluripotent stem cell (iPSC) lines. We assayed single-nucleus multiomics (snRNA/ATAC-seq) at baseline and after 1 h or 6 h of KCl stimulation. Results Analyzing expression QTLs (eQTLs) and chromatin accessibility QTLs (caQTLs), including allele-specific open chromatin (ASoC) variants, showed stronger enrichments for NPD GWAS risk among stimulation-specific QTLs than baseline QTLs, doubling the number of NPD risk loci that can be explained by functional risk variants. Integrating these QTLs with GWAS in a causal-transcriptome-wide association study (cTWAS) further identified putatively causal NPD risk genes predominantly under stimulated conditions. Notably, carnitine palmitoyltransferase 1C (CPT1C), a gene that facilitates lipid transport to mitochondria for fatty acid β-oxidation, was prioritized as a SZ causal gene only in stimulated excitatory neurons. Additionally, we found that SZ patients-derived neurons displayed a stronger induction of lipid/cholesterol synthesis genes (vs. control) only upon KCl stimulation. To establish the SZ causal role of CPT1C, we generated CRISPR-knockouts (KO) of CPT1C, together with another prioritized stimulation-specific SZ causal gene forkhead box N2 (FOXN2), in a iPSC-derived neuronal model. ScRNA-seq analysis showed that the differentially expression genes in CPT1C-KO and unexpectedly, FOXN2-KO excitatory neurons are strongly enriched for gene ontology terms related to lipid synthesis/metabolism, mitochondrial function, and neuronal function. Our high-content imaging analysis further confirmed that excitatory neurons of both CPT1C-KO and FOXN2-KO lines exhibited abnormal mitochondrial membrane potential and reactive oxygen species (ROS). Discussion Our study demonstrates that neuronal activation unmasks functional effects of many NPD risk variants and genes obscured in resting states or postmortem brains, revealing that SZ genetic risk genes partly converge on lipid-mitochondrial dysregulation.