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    Cellular and Molecular Bioengineering

    (I-337) Proline restores senescence-associated loss of mitochondrial function

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row I - Poster # 337
    Introduction:: Mitochondria, the dominant energy producing organelles of a cell, propels most biochemical reactions and are responsible for regulating metabolic activity and maintaining cellular homeostasis. Mitochondrial dysfunction has been widely regarded as one of the hallmarks of aging, as it drives cellular dysfunctions and worsens with age. Research in c-elegans or yeast models have shown that supplementation of a single amino acid, proline can promote longevity via balancing redox status. We wanted to extend those findings to mammalian cells and investigate the effect of proline on mitochondrial function in cellular senescence model.   

    Materials and Methods:: 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 DNA-damage (etoposide) induced senescence model. 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) and 14 days (S-pro-14).  

    Results, Conclusions, and Discussions:: Mitochondrial function was severely compromised in senescent MSCs (S), as was evidenced by significantly reduced mitochondrial membrane potential (MMP) and diminished mitochondrial respiratory capacity (Fig. 1A-C). This was accompanied by reduced mitophagy, the selective degradation of damaged mitochondria, and autophagic capacity. To understand the underlying molecular mechanism, RNA sequencing was performed and it was observed that the proline biosynthesis pathway was downregulated in aged cells which was confirmed via RT-PCR, Western Blot and measurement of intracellular proline content (Fig. 2A-C). Interestingly, treatment of senescent stem cells with proline restored mitochondrial function by inducing mitophagy leading to improved mitochondrial function and the reversal of aging hallmarks. Proline improved the mitochondrial function of the compromised senescent MSCs shown through restored MMP and mitochondrial respiratory activity (Fig. 1A-C). After 7 days of treatment, proline promoted mitophagy in senescent cells evidenced by upregulation of the PINK-PARKIN signaling pathway, staining of LC3 and Tomm20 (Fig. 3A-B) indicating that mitochondria in senescent cells were marked for degradation and restoration of autophagy capacity (Fig. 3C-D).  After 14 days, mitochondrial rejuvenation was found, including restoration of the myogenic capacity of senescent MSCs (Fig. 4A-B) and amelioration of aging hallmarks, such as reduced y-H2AX, Sa-B-gal, intracellular urea, and inflammatory cytokines. Improving mitochondrial function may be a preventative measure for many diseases stemming from abnormalities in the mitochondria and severe age-associated diseases, including encephalomyopathies, neurodegenerative diseases, cardiomyopathy, diabetes, and cancer. Our results suggests 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. 

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