Multiplexed Single Nucleus RNA and ATAC-seq Sequencing and Cortical Organoids: Transformative Insights into Down Syndrome - ABSTRACT A bold and risky approach is required to gain deep, transformative insights into the behavioral dysfunctions consequent to DS. A critical challenge in dissecting the effects and mechanisms of DS in brain function and ultimate appearance of DS-AD has been that the multitude of brain cell types and the connections between them impel a comprehensive approach that analysis of neuronal abnormalities, which can only be provided by contemporary single nucleus analyses that identify both transcripts and enhancers for each cell type in the CNS, which has been successfully applied in our laboratories to identify the transcriptional factors/pathways that underlie aging in each CNS cell type, and identify the distinct transcriptional/enhancer patterns that underlie the distinct trajectory tree associated with sporadic AD. Because DS causes abnormalities in early brain development, it is necessary to dissect the CNS consequences of DS both at early developmental times and with aging, corresponding to the time when AD appears. The challenge of solving the early developmental alterations in every cell type can be approached by using single nucleus ATAC-seq as a validation for predicted transcription factors and enhancers underlying AD in DS individuals, and distinguishing effects of the absence or presence of dementia, and by generating cortical organoids from control and DS iPSCs, with a deep, multiplexed analysis. This can be complemented by developing such cortical organoid cultures from DS iPSCs in which we have engineered a conditional silencing of HSA21 by using an Xist strategy, uncovering the specific temporal effects of HSA21 on brain cell functions. Because cortical organoid cultures do not generate oligodendrocyte precursors and do permit exploration of alterations with aging, we have extended the strategy by applying a similar multiplexed sc analysis in control and DS-model mice. This has the critical advantage of licensing a lineage tracing analysis. Such a multiplexed approach is actually likely to be transformative and to reveal many previously unanswered questions as to the origins of different classes of astrocytes and microglia, rather than neurons per se and the alternative choices in the route of development of oligodendrocyte precursor, both in normal neurogenesis and in DS vs sporadic AD.
