Abstract
Simple SummaryDown Syndrome, which is due to the presence of three copies of chromosome 21, always presents with mental retardation, possibly caused by defects in the development of neurons. In recent years, it has been shown that cells and tissues in Down syndrome manifest alterations in the function of mitochondria, the organelles that provide energy to cells. We hypothesized that mitochondrial dysfunction might contribute to the defect in neuronal cell development. To test this hypothesis, we generated a model of stem cells that, upon specific treatments, are capable of giving rise to neuronal cells, as evidenced by the synthesis of specific proteins. We observed that stem cells derived from Down syndrome individuals, after 21 days of growth in an artificial system, had an abnormal tendency to develop as glial cells, compared with control cells. As early as day 7 of culture, the trisomic cells also exhibited defects in mitochondrial function, such as anomalies in their calcium level, oxygen free radicals, oxygen consumption, and synthesis of ATP, a molecule that is critical in energy conversions. These results indicate that alterations in neuronal development and mitochondrial function occur early in this model, which we think is suitable for answering further questions.Background: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction. Methods: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer. Results: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. Conclusions: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.
Highlights
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21)
All these findings indicate that trisomy of Hsa21 (T21) affects mitochondrial function and, as a possible consequence, the production of reactive oxygen species (ROS) in neural tissues
In full agreement with the Quantitative real time polymerase chain reaction (qRT-PCR) data, we found that Eu-iPSCs and T21-iPSCs resulted positive for the stemness marker OCT4 and negative for the differentiation marker PAX6 (Figure 3A)
Summary
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa). The DS phenotype, which possibly results from the interaction between the over-expression of genes mapping to the trisomic chromosome and the subsequent dysregulation of genes mapping to different chromosomes, is always characterized by neuro-developmental anomalies and early neuro-degenerative processes [1].Impaired mitochondrial function in DS is widely documented. A decrease in mitochondrial membrane potential and ATP production, and an increase in reactive oxygen species (ROS) were demonstrated in the brain of a DS model, the Ts1Cje mouse [4]. Many studies in DS hippocampus and other tissues have reported aberrant hyperactivation of the AKT/mTOR signaling pathway suggesting that imbalances in autophagy flux regulation in DS leads to negative effects on mitochondrial turnover [7] All these findings indicate that trisomy of Hsa (T21) affects mitochondrial function and, as a possible consequence, the production of ROS in neural tissues. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation
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