Metabolic Reprogramming Underlying NANOG Induced Reversal of Aging Hallmarks

Introduction: Mitochondrial dysfunction, one of the major hallmarks of aging has been implicated in age-related loss of stem cell function and development of an inflammatory phenotype. Several cellular senescence models have shown deterioration of oxidative phosphorylation (OXPHOS), with senescent cells manifesting severe defects in respiratory chain complexes. In our study, we discovered that ectopic expression of the pluripotent transcription factor NANOG completely restored the mitochondrial function in senescent stem cells and that NANOG-driven rejuvenation led to metabolic reprogramming of senesced cells to the state of young proliferating cells.

Materials and

Methods: To study the effect of NANOG on aged cells, we employed several models of aging: replicative senescence of human Mesenchymal Stem Cells (MSC), myofibroblasts derived from patients suffering from Hutchinson’s Gilford Progeria Syndrome (HGPS), and progeric mice model (LAKI). Young MSCs (Y) were induced to senescence by serial passaging (S). Cells were transduced with tetracycline-regulatable lentivirus, which enabled NANOG expression by addition of doxycycline after cells reached senescence (SN).

Results, Conclusions, and Discussions: : Senescent cells (S) exhibited severe mitochondrial dysfunction, as was evidenced by significantly reduced mitochondrial membrane potential, cytochrome C oxidase (COX IV) protein expression (Fig A) and reactive oxygen species (ROS) accumulation. To delineate the cell metabolic profile, we employed Seahorse analyzer and observed complete restoration of mitochondrial respiratory chain capacity in aged MSCs after NANOG overexpression (Fig. B-C). Metabolomic study revealed increased dependence of senescent cells on glutamine to meet their bioenergetic demand, as confirmed by increased glutaminase (GLS) activity in S cells, which was suppressed by NANOG overexpression. This observation also held true for our in-vivo study, where we observed increased GLS expression and activity in the heart and skin (Fig. E-F) of progeria mice. Overactivated glutaminolysis coupled with loss of urea transporter, SLC14A1 resulted in accumulation of toxic end-product, urea leading to detrimental consequences for mitochondrial function and DNA damage. In addition, we observed that GLS1 expression was regulated through one of the MAPK (stress) pathways namely, the JNK pathway in senescent cells. Interestingly, inhibition of GLS activity in senescent cells via CB-839 (S-CB) decreased urea accumulation, partially restored mitochondrial function (Fig. B-C), decreased ROS and DNA damage. Furthermore, we examined the effects of CB-839 on LAKI mice and observed decreased age-associated ROS and urea accumulation in heart, skin and muscle, concomitant with improved mitochondrial function (Fig. G-H). Geriatric assessment tools were employed to assess the overall health of progeria mice after CB-839 treatment. We observed that improved mitochondrial function in progeria mice was associated with improved gait speed, inverted hanging time, increased ambulatory speed and distance and improved ability to build their nest, suggesting overall improved health with glutaminolysis inhibition.

Acknowledgements (Optional):