HD (Huntington's disease) is a devastating neurodegenerative genetic disorder caused by abnormal expansion of CAG repeats in the HTT (huntingtin) gene. We have recently established two iPSC (induced pluripotent stem cell) lines derived from a HD patient carrying 72 CAG repeats (HD-iPSC). In order to understand the proteomic profiles of HD-iPSCs, we have performed comparative proteomic analysis among normal hESCs (human embryonic stem cells; H9), iPSCs (551-8) and HD-iPSCs at undifferentiated stages, and identified 26 up- and down-regulated proteins. Interestingly, these differentially expressed proteins are known to be involved in different biological processes, such as oxidative stress, programmed cell death and cellular oxygen-associated proteins. Among them, we found that oxidative stress-related proteins, such as SOD1 (superoxide dismutase 1) and Prx (peroxiredoxin) families are particularly affected in HD-iPSCs, implying that HD-iPSCs are highly susceptible to oxidative stress. We also found that BTF3 (basic transcription factor 3) is up-regulated in HD-iPSCs, which leads to the induction of ATM (ataxia telangiectasia mutated), followed by activation of the p53-mediated apoptotic pathway. In addition, we observed that the expression of cytoskeleton-associated proteins was significantly reduced in HD-iPSCs, implying that neuronal differentiation was also affected. Taken together, these results demonstrate that HD-iPSCs can provide a unique cellular disease model system to understand the pathogenesis and neurodegeneration mechanisms in HD, and the identified proteins from the present study may serve as potential targets for developing future HD therapeutics.
Redox balance has been suggested as an important determinant of "stemness" in embryonic stem cells (ESCs). In this study, we demonstrate that peroxiredoxin (Prx) plays a pivotal role in maintenance of ESC stemness during neurogenesis through suppression of reactive oxygen species (ROS)-sensitive signaling. During neurogenesis, Prx I and Oct4 are expressed in a mutually dependent manner and their expression is abruptly downregulated by an excess of ROS. Thus, in Prx I 2/2 or Prx II 2/2 ESCs, rapid loss of stemness can occur due to spontaneous ROS overload, leading to their active commitment into neurons; however, stemness is restored by the addition of an antioxidant or an inhibitor of c-Jun N-terminal kinase (JNK). In addition, Prx I and Prx II appear to have a tight association with the mechanism underlying the protection of ESC stemness in developing teratomas. These results suggest that Prx functions as a protector of ESC stemness by opposing ROS/JNK cascades during neurogenesis. Therefore, our findings have important implications for understanding of maintenance of ESC stemness through involvement of antioxidant enzymes and may lead to development of an alternative stem cell-based therapeutic strategy for production of high-quality neurons in large quantity. STEM CELLS 2014;32:998-1011
Key Points• OVOL2 is identified as a novel binding protein of ER71.
• Interaction between ER71and OVOL2 cooperatively regulates the generation of FLK1 1 mesoderm, and endothelial and erythroid cells.In this study, we report that OVOL2, a C 2 H 2 zinc finger protein, is a novel binding protein of ER71, which is a critical transcription factor for blood and vessel development. OVOL2 directly interacted with ER71, but not with ETS1 or ETS2, in the nucleus. ER71-mediated activation of the Flk1 promoter was further enhanced by OVOL2, although OVOL2 alone failed to activate it. Consistently, coexpression of ER71 and OVOL2 in differentiating embryonic stem cells led to a significant augmentation of FLK1 1 , endothelial, and hematopoietic cells. Such cooperative effects were impaired by the short hairpin RNA-mediated inhibition of Ovol2. Collectively, we show that ER71 directly interacts with OVOL2 and that such interaction is critical for FLK11 cell generation and their differentiation into downstream cell lineages. (Blood.
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