The role of polyamines in different models of senescence

Resumen

Introduction

Aging is a complex biological process characterized by the progressive accumulation of cellular and molecular damage, associated with structural and functional changes that promote the development of age-related diseases. One of the central mechanisms involved is cellular senescence, a state of irreversible cell cycle arrest accompanied by morphological, metabolic, and molecular changes, including increased senescence-associated β-galactosidase (SA-β-gal) activity, decreased Lamin B1, and epigenetic alterations such as changes in H3K9me3.  In addition to these changes, polyamines (PAs) are directly linked to cellular senescence through their role in metabolic pathways such as protein biosynthesis, mitochondrial integrity, and immune system regulation. PAs, particularly putrescine, spermidine (the most studied), and spermine, are small polycationic molecules at physiological pH that participate in processes essential for cellular homeostasis.  Polyamine levels decline progressively with age, contributing to alterations in cellular function; however, their specific role in the establishment and maintenance of the senescent phenotype remains to be fully understood.

Objective

To evaluate the role of the polyamines spermidine, spermine, and putrescine in different models of induced cellular senescence, physiological aging, and premature aging (Hutchinson-Gilford Progeria Syndrome, HGPS).

Methodology

Primary human fibroblasts corresponding to three cellular models will be used: physiological aging (from an elderly donor), premature aging (from an individual with HGPS), and a model of senescence induced by hydrogen peroxide. The senescent phenotype will be characterized by SA-β-gal staining, Lamin B1 assessment, analysis of H3K9me3 and γH2Ax marks, and analysis of cytoskeletal organization. Total polyamine content and individual levels of putrescine, spermidine, and spermine will be quantified by fluorometric assays and high-performance liquid chromatography (HPLC). Furthermore, the expression and subcellular localization of ornithine decarboxylase (ODC-1) will be evaluated. Finally, the effect of exogenous polyamine supplementation on senescence markers and cell viability will be analyzed.

Results

Our results confirmed the establishment of cellular senescence in the different fibroblast models through the evaluation of classical senescence markers, including SA-β-gal activity, Lamin B1, H3K9me3, and γH2AX. Furthermore, total polyamine levels were significantly altered in senescent cultures, including induced, physiologically aged, and prematurely aged fibroblasts, compared with control cells. HPLC analyses revealed that spermidine (SPD) levels were significantly reduced across all senescent models compared with control cultures. Based on these findings, senescent cultures were supplemented with exogenous SPD, resulting in a significant attenuation of senescence-associated markers and a phenotype comparable to that of control cultures. In addition, alterations in key autophagy-related proteins were observed in senescent cultures and were partially restored following SPD supplementation.

Conclusions

Our findings indicate that spermidine attenuates key hallmarks of the senescent phenotype, irrespective of the mechanism by which senescence was induced. Furthermore, these effects may be mediated through the modulation of autophagic flux, a process closely linked to aging and cellular senescence.