SINGLE-CELL ANALYSIS IDENTIFIED CELL-TYPE-SPECIFIC AND CONVERGED PATHWAYS OF AUTISM SPECTRUM DISORDER

Abstract Patients with psychiatric disorders such as autism spectrum disorder (ASD) showed various and heterogeneous clinical symptoms even share the same mutation. Furthermore, the comorbidity of psychiatric disorders makes it more complicated, despite of clinical manifestations of ASD that have been established. In genetic and genomic aspects, ASD has been categorized as a genetic disorder due to studies investigating concordant and discordant monozygotic twins. These past genetic studies have also identified chromosome mutations such as deletion and duplication larger than 1 kb in patients with ASD. These chromosome abnormalities named copy number variations (CNVs) are one of the risk factors associated with ASD. Here, we applied single-cell RNA sequencing (scRNA-seq) to identify CNV- and cell- type-specific abnormalities in ASD by using differentiated neurons from genetically edited mouse embryonic stem (ES) cells. Each mutant cell line used in this study has a psychiatric disorder associated CNV such as 1q21 duplication, 2p16.3 deletion, 3q29 deletion, 7q11.23 duplication, 15q11 deletion, 15q13.3 deletion, 16p11.2 deletion, 16p13.2 deletion, 17p11.2 deletion, 17q12 deletion, Xq27 (Fmr1) deletion, and Xq28 (Mecp2) deletion. Uniform Manifold Approximation and Projection (UMAP), the mathematical manifold learning technique for dimension reduction, identified 16 cell types such as glutamatergic and GABAergic neurons, neural stem cells, microglia, oligodendrocyte precursor cells, and endothelial cells. These cell types were annotated according to cell-type markers. Overall, network analysis based on the differentially expressed gene (DEGs) identified enriched terms such as “splicing”, “neural system”, “axon guidance”, “mitochondria”, and “translation”. Interestingly, “immune system” was also a significant term in these CNVs. We also found a unique association between “endoplasmic reticulum stress signaling” and “GABAergic neurons”, as well as “p53 signaling” and “microglia” respectively in pathway analysis. Surprisingly, “p53 signaling” was preferentially affected by “microglial cluster” regardless of CNV type. Lastly, we focused on “translation machinery” because previous studies have highlighted this biological process as a converged pathway of ASD. Through our transcriptome analysis, we also identified “translation” as a commonly dysregulated pathway regardless of CNV- and cell types. In particular, the mTORC1 complex which includes mTOR is affected in neurons. In conclusion, our comprehensive transcriptome analysis in single-cell resolution emphasizes not only molecules, and networks, but also converged pathways of ASD which may contribute to the drug development of ASD. References Nomura, J. et al. (2022) Autism in a dish: Es cell models of autism with copy number variations reveal cell-type-specific vulnerability [Preprint]. doi:10.1101/2022.02.02.478766.