Investigation of the intracellular and extracellular mechanisms underlying the process of aging

National Institute On Aging

Investigators

Abstract

Despite established studies on EVs in chronic aging, their roles in mediating telomere shortening-induced biological defects are largely unexplored. Furthermore, it is unclear whether EVs are affected by telomere shortening, which is a defining characteristic of aging. In this study, we have explored these questions, with a focus on circulating plasma and tissue EVs derived from a human aging cohort and a telomerase knockout mouse model. We have demonstrated that the human aging cohort exhibited altered EV levels and telomeric DNA cargo. We conducted an in-depth analysis of EVs from plasma and six different tissues (brain, heart, liver, muscle, spleen, and small intestine) using the telomerase reverse transcriptase knockout (Tert-/-) mouse, which is deficient in telomerase and undergoes progressive telomere shortening across generations while recapitulating features of aging, age-related diseases, and telomere biology disorders based on their relative proliferative capacity. The following is a concise summary of our work: 1) We have characterized the concentration, size, and telomeric DNA and protein cargos in EVs derived from WT and telomerase-deficient mice, as well as in a human aging cohort. Our findings underscore the intricate diversity among plasma- and tissue-derived EV dynamics. Interestingly, the alterations in EV concentration and telomeric DNA cargos predominantly observed in telomerase-deficient mice with shortened telomeres resemble those observed in the human aging cohort. In addition, we uncovered a correlation between the generation of EV telomeric DNA cargos and rapid proliferation, replication stress, and telomere-end deprotection. Our proteomic analysis revealed significant changes in EV protein cargos from telomerase-deficient mice with shortened telomeres, offering the potential for future biomarker assessment in telomere shortening-associated aging and disease conditions. 2) Our functional assays showed that EVs derived from telomerase-deficient mice with shortened telomeres adversely affected recipient cells. Firstly, we examined the inflammatory response in bone marrow-derived macrophages (BMDMs) treated with plasma EVs. BMDMs exposed to EVs from G3 Tert-/- mice exhibited a significant increase in IL-6 levels, indicating a pro-inflammatory response, consistent with previous studies linking telomere loss to chronic low-grade inflammation, a hallmark of aging-related pathologies. In addition, a slight elevation in CCL2 expression suggests a role for EV-mediated communication in chemotactic responses associated with telomere shortening. Furthermore, our cytokine array analysis provided a comprehensive view of the inflammatory milieu induced by EV treatment in BMDMs. Despite there being a mild overall response observed in BMDMs, the significant upregulation of IFNγ, IL2, IL7, CXCL9, and CCL12 highlights the complex network of cytokine and chemokine signaling pathways modulated by EVs from G3 Tert-/- mice. Our data underscore the robustness of our findings and implicate the potential role of EVs as mediators of the inflammatory response associated with telomere shortening. Secondly, we evaluated neurotoxicity in primary cultured neurons following EV treatment, since mice with short telomeres display neurodegeneration. Neurons treated with EVs from G3 Tert-/- mice showed a slight decrease in survival compared to those treated with EVs from WT mice, but there was no noticeable difference in neurons that received brain-derived EVs. These findings suggest tissue-specific variations in EV-mediated effects on neuronal viability, illustrating the importance of considering both the cellular source and recipient context in interpreting EV-mediated signaling outcomes. Overall, our study underscores the multifaceted role of EV-mediated communication in regulating inflammatory responses and neurotoxicity in the context of telomere shortening. Understanding the underlying molecular mechanisms and identifying key mediators in EV-mediated signaling pathways hold promise for the development of targeted therapeutic interventions to mitigate pathologies associated with short telomeres. The work was published (iScience, 2025, 28:112661. PMCID: PMC12158500. PMID: 40502707).