c-Jun N-Terminal Kinase Inhibition Induces Mitochondrial Oxidative Stress and Decreases Survival in Human Neural Stem Progenitors

Abstract

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.

Keywords: Apoptosis/viability/proliferation; Forkhead homeobox type O3a; Mitochondrial membrane potential; Mitochondrial superoxide; Neural stem cells; Stress activated kinases/c-Jun N-terminal kinases.