Neural stem cells are attracting enormous attention in regenerative medicine due to their ability to self-renew and differentiate into the cell lineages that constitute the central nervous system. However, little is known about the mechanism underlying the regulation of their redox environment, which is essential for homeostatic cellular functions. The redox-modulated c-Jun N-terminal kinases (JNK) are a molecular switch in stress signal transduction and are involved in numerous brain functions. Using a selective but broad-spectrum inhibitor of JNK 1/2/3, we investigated the role of JNK in regulating the levels of reactive oxygen species in mitochondria, mitochondrial membrane potential, viability, proliferation and lineage alterations in human H9-derived neural stem/progenitor cells (NSPs). Relative to diluent control, incubation of the NSPs for 24 h with SP600125, an anthrapyrazolone inhibitor of JNK, resulted in increased abundance of mitochondrial superoxide radicals (p < 0.05), concomitant with decreases in mitochondrial membrane potential (p < 0.001), while maintaining a consistent and stable mitochondrial mass. Whereas H9-derived NSPs collectively express Nestin, a marker for neural stem cells, a panel of cell surface markers analyzed by flow cytometry revealed that they are a heterogeneous population that sustains this diversity after JNK inhibition. In addition, the levels of nuclear forkhead homeobox type O3a (FoxO3a), a regulator of redox homeostasis, decreased, which was associated with a decrease in overall cell viability as measured by Annexin V staining (p < 0.001), and supported by an increased level of cleaved Poly-ADP-ribose polymerase and decreased survivin expression. However, staining with the proliferation marker, Ki67, revealed the presence of a significant percentage of proliferating cells in the treated population. Together, the results support a role for JNK in the redox-homeostasis and fate of NSPs. Identifying regulators of the cellular redox environment will enhance our understanding of the mechanisms that modulate neural stem cell functions and optimize therapeutic applications targeting JNK.

中文翻译：

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c-Jun N末端激酶抑制作用诱导线粒体氧化应激并降低人类神经干祖细胞的存活率。

神经干细胞具有自我更新和分化成构成中枢神经系统的细胞谱系的能力，因此在再生医学中引起了极大的关注。然而，关于其氧化还原环境调节的基础机制知之甚少，这对于稳态细胞功能至关重要。氧化还原调节的c-Jun N末端激酶（JNK）是应激信号转导中的分子开关，参与许多脑功能。使用选择性但广谱的JNK 1/2/3抑制剂，我们研究了JNK在调节人H9衍生神经干中线粒体中活性氧水平，线粒体膜电位，活力，增殖和谱系变化中的作用/祖细胞（NSP）。相对于稀释剂控制，将NSP与JNK的吡唑啉酮抑制剂SP600125孵育24小时，导致线粒体超氧化物自由基的丰度增加（p <0.05），同时线粒体膜电位降低（p <0.001），同时保持一致且稳定的线粒体质量 源自H9的NSP共同表达神经干细胞的标志物Nestin，而通过流式细胞仪分析的一组细胞表面标志物显示，它们是异质性种群，在JNK抑制后仍维持这种多样性。此外，氧化还原稳态的调节剂，O3a型核叉头同源盒（FoxO3a）的水平降低，这与膜联蛋白V染色测得的总体细胞活力降低有关（p <0.001），并受到裂解的Poly-ADP-核糖聚合酶水平提高和survivin表达降低的支持。然而，用增殖标记Ki67染色显示在治疗的人群中存在大量百分比的增殖细胞。总之，这些结果支持了JNK在NSP的氧化还原稳态和命运中的作用。识别细胞氧化还原环境的调节剂将增进我们对调节神经干细胞功能并优化针对JNK的治疗应用的机制的了解。