Inhibition of Glutaminolysis Restores Age-associated Loss of Mitochondrial Function

Introduction:: Introduction: Mitochondrial dysfunction, a major hallmark of aging, has been implicated in age-related loss of stem cell function and development of an inflammatory phenotype in aging individuals. Several cellular senescence models have shown deterioration of mitochondrial oxidative phosphorylation (OXPHOS), with senescent cells manifesting severe defects in respiratory chain complexes. Our study aimed to investigate the effect of NANOG, a pluripotent transcription factor, on cellular senescence. Our findings indicate that ectopic expression of NANOG rewires cellular metabolic pathways, resulting in complete restoration of mitochondrial function in senescent stem cells. Furthermore, our study showed that NANOG-driven rejuvenation led to metabolic reprogramming of senescent cells to a state similar to that of young proliferating cells.


Materials and Methods:: Materials and Methods: In our study, we employed several models of aging, including replicative senescence of human Mesenchymal Stem Cells (MSC), myofibroblasts from patients with Hutchinson’s Gilford Progeria Syndrome (HGPS) and a mice model of accelerated aging i.e., progeria mice model (LAKI). For our in-vitro studies, young, proliferating MSCs (Y) were induced to senescence by serial passaging (S). To study the effect of NANOG on aged cells, cells were transduced with tetracycline-regulatable lentivirus, which enabled NANOG expression by addition of doxycycline after cells reached senescence (SN). To study the effect of GLS1 inhibition in progeria mice, LAKI mice were injected with GLS1 inhibitor, CB-839 intraperitoneally, thrice a week for a period of 1 month (LAKI-CB).


Results, Conclusions, and Discussions:: Results and Discussion: 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 glutaminolyis inhibition.

Conclusions: 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 for restoring age-associated loss of mitochondrial function.


Acknowledgements (Optional): : This work was supported by grants from the National Institutes of Health, R01AG052387, R01AG068250 and from NYSTEM (Contract #C30290GG) to Stelios T. Andreadis.


References (Optional): : Choudhury, D., Rong, N., Ikhapoh, I., Rajabian, N., Tseropoulos, G., Wu, Y., ... & Andreadis, S. T. (2022). Inhibition of glutaminolysis restores mitochondrial function in senescent stem cells. Cell reports, 41(9), 111744

Shahini, A., Choudhury, D., Asmani, M., Zhao, R., Lei, P., & Andreadis, S. T. (2018). NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings. Stem Cell Research, 26, 55-66

Shahini, A., Rajabian, N., Choudhury, D., Shahini, S., Vydiam, K., Nguyen, T., ... & Andreadis, S. T. (2021). Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo. Science Advances, 7(36), eabe5671