Multi-omics approach in Down syndrome resolves new regulators in two regions of the human brain

Down syndrome (DS) is the most common genetic form of intellectual disability. DS is a very complex genetic condition and is caused by trisomy of human chromosome 21. Studies mostly on human DS fetuses, plasma, fibroblasts, iPSCs, and mouse models of DS have demonstrated genome-wide dysregulation in genes and proteins involved in brain development and proteostasis. However, these studies were obtained with low-throughput technologies and addressed only either gene or protein level. Thus, it is still unknown how the gene dysregulation relates to protein dysregulation and, in turn, to DS brain deficits. Here, taking advantage of high throughput technologies, we explored in parallel both gene and protein levels on the same postmortem tissue from DS and age/sex-matched control individuals. We have revealed shared patterns of the transcriptome at gene and transcript levels and proteome dysregulation in hippocampus and cortex of DS individuals. We found many non- triplicated genes and proteins differentially expressed along with overexpression in most of the triplicated genes and proteins. We identified many dysregulated biological processes in both brain regions, such as translation, axon development, synaptic signaling, neuron development, and mRNA splicing. We also found differentially expressed genes and proteins involved in extracellular vesicles and cell-substrate junction among all the cellular components identified in our study. In particular, our work highlights downregulation of neuronal genes together with downregulation of neuron-specific pathways such as long term potentiation (LTP) and synaptic vesicle cycle, and upregulation of microglia and astrocytic genes together with the increased response of inflammatory pathways such as complement and coagulation cascades and cytokine-cytokine receptor interaction, as key points in DS in both regions at gene and protein levels. Furthermore, we observed an alteration in RNA splicing in DS brains, which was shared across brain regions and involved many neurodevelopmental genes belonging to axon formation and guidance, dendrite morphogenesis, neurogenesis, and synaptic signaling. We show that genes related to axon formation and one of the RNA binding protein (PTBP2) that regulate these genes are differentially expressed and have differential splicing in DS brains. Interestingly, we collected the first experimental evidence (in murine neurons) of deficit in axonal polarization (multiple axon formation), establishing it as a definite phenotypic defect in DS. Finally, we performed small RNA sequencing on these postmortem human hippocampi and cortex and found several differentially expressed miRNAs, which provides information about another layer of gene- regulation in DS. Our findings indicate genome-wide dysregulation in adult DS hippocampus and cortex in a comprehensive assessment of both transcriptome and proteome. These data provide a unique and extensive resource for the field of DS. They will likely prove critical in identifying genetic drivers (among triplicated but also non-triplicated genes) to regulate complete biological systems at organelle-, cell-, process- or tissue-level to better understand the impact of trisomy 21 on people with DS, and thus derive future therapeutics.