Poster V10 - Proline ameliorates senescence hallmarks and restores mitochondrial function via activation of autophagy

Introduction: 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.

Materials and

Methods: To study the effect of proline on aged or senescent cells, we employed three widely established models of aging: replicative senescence of human Mesenchymal Stromal Cells (MSC), an accelerated aging mouse model ie. the progeria mouse model (Lamin-A Knock In, LAKI) and naturally aging mice (22-26 months). Young MSCs (Y) were induced to senescence by serial passaging (S). For our in vitro experiments, senescent MSCs were treated with 4mM proline for 7 days (S-pro-7) or 14 days (S-Pro-14). For our in-vivo system, LAKI mice and aged mice were fed with 5% w/v proline in drinking water for 2 months.

Results, Conclusions, and Discussions: Mitochondrial function was severely compromised in senescent MSCs (S), as was evidenced by significantly reduced mitochondrial respiratory capacity. This was accompanied by reduced 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 that the rate-limiting enzyme in the proline biosynthesis pathway (PYCR1) to be significantly downregulated in senescent cells, further confirmed by reduced PYCR1 protein expression in S cells (Fig. 1A), as well as reduced intracellular proline content (Fig. 1B). This observation held true for our in-vivo study, where we observed decreased PYCR1 expression in the skin tissue of aged mice. Interestingly, treating the senescent mesenchymal stromal cells with proline for 14 days (S-pro-14) not only improved mitochondrial function (Fig. 2A) but also increased the protein expression of mitochondrial electron transport chain complexes (Fig. 2B). Surprisingly, short-term treatment of proline for 7 days (S-pro-7) did not restore mitochondrial function (Fig. 2A) which led to our hypothesis that initially, proline may increase mitophagy after 7 days to get rid of damaged mitochondria, eventually leading to improved mitochondrial function after 14 days. Indeed, 7 days of proline induced mitophagy by activating AMPKα (Fig. 3A), thereby upregulating Parkin expression (Fig. 3B), the major regulators in mitophagy process. Furthermore, we employed mt-Keima, a pH-sensitive fluorescent probe that enables distinction of mitochondria in the acidic (autolysosomes) or neutral pH environment based on excitation at 560nm and 440nm respectively. Our results indicated marked increase in acidic mitochondria in S-pro-7 cells indicating an elevated mitophagy flux (Fig. 3C). Restoration of mitochondrial function by proline treatment was accompanied by amelioration of several aging hallmarks like DNA damage (Fig. 4A), SA-βGal (Fig. 4B) and inflammatory cytokine production. Currently we are investigating the effects on heart and skin tissue of aged mice to assess the translatability of our in-vitro findings to an in-vivo context.
In summary, we report that proline treatment restored mitochondrial function, which was impaired by cellular aging, suggesting novel strategies to reverse or delay cellular senescence, with broad applications in tissue regeneration and potentially anti-aging treatments.

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