Bioinformatic and functional analyses of REST dysregulation in human iPSC-derived neural progenitors, astrocytes, and organoids in Down syndrome

This study aimed to confirm RE1-silencing transcription factor (REST)-mediated molecular dysregulation in Down syndrome (DS) brains and neural cells. To explore the role of REST in fate determination in trisomic neural progenitor cells and the reactivity of trisomic astrocytes. This study first analysed transcriptomic data from DS-derived brain tissues, cerebral organoids, neural progenitor cells (NPCs), neurons, and astrocytes to identify differentially expressed genes (DEGs) and determine REST- targeted DEGs using R software and the Galaxy web platform. REST-targeted DEGs were enriched across multiple brain regions, ages, and neural cell types in various signalling pathways, including JAK-STAT, neural cell differentiation, inflammation, neural cell function, and metabolic homeostasis. Further analysis of DS human induced pluripotent stem cell (hiPSC)-derived NPC transcriptomic data revealed that REST-targeted DEGs were associated with neural cell differentiation and JAK-STAT pathway dysregulation. RT-qPCR validation showed the upregulation of all five REST-targeted hub genes (CD44, TGFB1, FN1, ITGB1, and COL1A1), JAK2, and STAT3, along with the NFIA (a glioblast marker) upregulation and DCX (a neuroblast marker) downregulation. Western blot and Immunocytochemistry (ICC) identified the DCX (a neuroblast marker) downregulation and NFIA (a glioblast marker) upregulation. Thus, REST downregulation may be critical to the neurogenic-to- gliogenic shift observed in trisomic NPCs. I further analysed transcriptomic data from DS hiPSC-derived cerebral organoids at different development stages (30-day to 160- day organoids). REST-targeted DEGs in 90-day cerebral organoids were significantly enriched in the JAK-STAT signalling pathway and gliogenesis, distinct from other time points. RT-qPCR validation confirmed the upregulation of six REST-targeted hub genes (CSTB, MCM3AP, PFKL, POFUT2, PRMT2 and RWDD2B) and STAT3 in 90-day cerebral organoids, along with the downregulation of DCX and upregulation of NFIA. These findings identified that the loss of REST is a critical factor in the neurogenic-to-gliogenic shift during DS brain development. To further investigate the function of increased astrocytes in DS, R software was used to analyse transcriptomic data of DS hiPSC-derived astrocytes. REST-targeted DEGs were related to cell differentiation, neuroinflammation, and lipid metabolism. RT-qPCR and ICC identified the REST loss in trisomic astrocytes compared with controls. RT-qPCR further validated that the loss of REST led to the upregulation of inflammatory cytokines (IL-10, IL-1B, TNF-α, and TGF -β) and reactive astrocyte markers (EAAT2, GFAP, S100A10, and S100B). These gene expression patterns in disomic hiPSC- derived astrocytes treated with X5050 (100μM, REST-degraded) were like those in trisomic astrocytes. However, trisomic hiPSC-derived astrocytes with lithium carbonate treatment (5mM, REST-restored) repressed inflammatory cytokine and reactive astrocyte markers expression. Thus, REST loss is vital to trisomic astrocyte reactivity. Reactive astrocytes may have dual functions (neurotoxic and neuroprotective) in DS, which warrants further investigation. This study lays the foundation for a better understanding of the role of REST in contributing to DS neuropathology by using the trisomic hiPSC-derived cerebral organoids, NPCs, and astrocytes. REST should be an ideal candidate as one of the therapeutic targets for DS.

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