(471a) Mitochondrial Mechanisms Underlying NANOG Induced Reversal of Aging

Mitochondrial dysfunction, one of the major hallmarks of aging has been implicated in age-related loss of stem cell function and development of 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 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.

To study the effect of NANOG on aged cells, we employed several models of aging i.e., 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 were induced to senescence by serial passaging. Cells were transduced with tetracycline-regulatable lentivirus, which enabled NANOG expression by addition of doxycycline, after cells reached senescence.

Senescent or aged cells exhibited severe mitochondrial dysfunction, as was evidenced by significantly reduced mitochondrial membrane potential, reduced respiratory function and excessive accumulation of reactive oxygen species (ROS). To delineate cell metabolic profile, we employed Seahorse analyzer and observed complete restoration of mitochondrial respiratory capacity in aged MSCs after NANOG overexpression. Metabolomics study revealed increased dependance of senescent cells on glutamine to meet their bioenergetic demand and this was confirmed by increased glutaminase (GLS) activity in aged cells. This observation also held true for our in-vivo study, where we observed increased GLS expression and activity in heart, aorta and skin of progeric (LAKI) as well as geriatric mice. Enhanced deamination of glutamine with senescence results in accumulation of toxic end-product, urea leading to detrimental consequences for mitochondrial function and DNA damage. Interestingly, inhibition of GLS activity in senescent cells by CB-839 decreased urea accumulation and partially restored mitochondrial function, 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. Hence, inhibition of GLS activity rejuvenated mitochondrial function and led to amelioration of aging hallmarks.

In conclusion, our data provide novel insight into the mechanism, underlying metabolic reprogramming associated with cellular aging. Targeting glutamine metabolism and associated metabolites and byproducts may be a promising strategy to delay or reverse cellular senescence.