| 其他摘要 | Background: Type-I bipolar disorder (BDI) is a chronic, complex psychiatric disorder with an incidence of ~0.6-1.0%, and is characterized by severe mania with or without depressive onset. Although the lithium therapy is effective for a majority of patients with BDI, however, approximately 30-40% of patients fail to show positive response to lithium. The development of single-cell RNA sequencing (scRNAseq) technique has made it possible to identify cell type-specific pathophysiological changes of diseases. Due to the difficulties in directly analyzing the human brain, brain organoids derived from induced pluripotent stem cells (iPSCs) of patients have been applied to disease research. Due to their merits of enriched cell types, organizational structure, and reproducible cell-to-cell interaction and signal transduction, brain organoids differentiated from patient iPSCs provide a powerful tool for the investigation of the pathophysiology of diseases.
Objective: To explore the mid-brain pathogenic mechanisms of BDI and mechanisms of lithium response difference by constructing the patient iPSC-derived midbrain organoid disease model and performing RNAseq and scRNAseq analyses.
Methods: 1) Differentiation of BDI patients and health people derived iPSCs into midbrain organoids by adding the TGFβ signaling pathway inhibitor SB431542, the BMP signaling pathway inhibitor LDN193189, the SHH signaling pathway activators SHH and FGF8, and the WNT signaling pathway activator CHIR99021, to induce the formation of the neural floor plate and neurogenesis, and finally adding TGFβ, cAMP, BDNF and GDNF to promote the maturation of dopaminergic neurons and the midbrain organoids. 2) RNAseq and single-cell RNAseq analyses of human midbrain organoids to assess the proportion of cell types and the cell cycle.
Results: 1) The total transcriptome analysis revealed that there were significant differences between the midbrain organoids of patients and healthy controls, and there were 1615 differentially expressed genes (DEG) between the lithium responsive (LR) roup and the healthy human (HC) group. The lithium non-responsive (NR) group had a total of 2969 DEGs compared to the HC group. There were a total of 2859 DEGs between the LR group and the NR group. 2) The total transcriptome analysis revealed that compared to the HC group, the LR group showed a significant dysfunction in the synaptic pathway, the calcium signaling pathway and the neuroactive ligand-receptor interaction pathway, and the NR group showed a significant dysfunction in the glycerol phospholipid metabolic pathway, the steroid biosynthetic pathway and the amino sugar and nucleotide glucose metabolic pathway. 3) A total of 28389 cells were obtained for single-cell downstream analysis after quality control, and were classified into seven cell types, including dopaminergic neurons, astrocytes, oligodendrocytes, neural progenitor cells, oligodendrocyte progenitor cells, radial glial cells and endothelial cells. 4) In BDI patient iPSC-derived midbrain organoids, the number of neurons especially the dopaminergic neurons were significantly increased, the number of glial cells especially the oligodendrocytes were significantly decreased and the number of astrocytes was increased. 5) The expression of PLD3 were downregulated in the dopaminergic neurons and astrocytes of BDI patient iPSC-derived midbrain organoids, especially in the NR group.
Conclusion: 1) BDI midbrain organoids disease models were successfully constructed. 2) The gene expression differed significantly between the midbrain organoids of the BDI patients and healthy control, and the total transcriptome of the LR and Multiple synaptic-related pathways, calcium Signaling pathway, circadian entertainment, cAMP signaling pathway, glucose metabolism, lipid metabolism and amino acid metabolism-related pathways. Lithium therapy mainly works by regulating synaptic related pathways, calcium signaling and circadian rhythm. 3) In the BDI patient organoids, blocked cell proliferation promoted iPSCs differentiation into DA neurons, which might represent a manic behavioral characterization. 4) The decrease of oligodendrocyte number may be one of the pathogeneses of BDI. 5) The downregulation of PLD3 expression may be one of the reasons for lithium nonresponse. |
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