(511b) Proline Restores Mitochondrial Function and Reverses Aging Hallmarks in Aged Stem Cells

Mitochondria, the major energy producing organelles of a cell perform a myriad of biochemical reactions and play vital roles in cellular stress responses and tissue homeostasis. Selective degradation of damaged mitochondria via mitophagy is crucial for maintenance of healthy mitochondrial pool. Accumulation of dysfunctional mitochondria is one of the major hallmarks of aging and has been implicated as a causal factor in driving the aging phenotype. In our study, we discovered that treatment of senescent or aged stem cells with the amino acid, proline restored mitochondrial function via activating mitophagy, and proline driven mitophagy led to reversal of aging hallmarks.

To study the effect of proline on aged cells, we employed two widely established models of aging: replicative senescence of human Mesenchymal Stem Cells (MSC) and a progeria mouse model (Lamin-A Knock In, LAKI). Young MSCs (Y) were induced to senescence by serial passaging (S). For our in vitro experiments, senescent MSCs were treated with 4mM proline for 2 weeks (S-Pro). For our in-vivo system, LAKI mice were fed with 5% w/v proline in drinking water for 2 months.

Mitochondrial function was severely compromised in senescent MSCs (S), as was evidenced by significantly reduced mitochondrial respiratory capacity. This was accompanied by reduced mitophagy and autophagic capacity of aged cells, leading to accumulation of defective and depolarized mitochondria. To explore the underlying molecular mechanism, we performed RNA sequencing analysis and observed the proline cycle gene (PYCR1) to be significantly downregulated in senescent cells, further confirmed by reduced PYCR1 protein expression in S cells. This observation held true for our in-vivo study, where we observed decreased PYCR1 expression in heart and skin tissue of aged mice as well as decreased proline content. Interestingly, proline treatment significantly improved the mitochondrial function of aged cells. Proline increased the phosphorylation of AMPKα, thereby activating the PINK-PARKIN signaling pathway, upregulating mitophagy, and resulting in enhanced clearance of depolarized mitochondria. Restoration of mitochondrial function by proline treatment was accompanied by amelioration of several aging hallmarks like DNA damage, SA-βGal and inflammatory cytokine production. In addition, evaluation of heart and skin tissue of proline fed progeric mice, demonstrated significantly improved mitochondrial function and decreased ROS accumulation, suggesting that proline supplementation might provide a viable approach in mitigating age-related mitochondrial impairment.

We report that proline treatment restored mitochondrial function, which was impaired by cellular aging, and suggests novel strategies to reverse or delay cellular senescence, with broad applications in tissue regeneration and potentially anti-aging treatments.