Biology subjects

Carosi, J. M.

Publications and source records attributed to Carosi, J. M..

3 recordsLinked to original sources

bioRxiv2025

SenLect: a genetically encoded system to purify senescent cells

Cellular senescence is a state of irreversible cell cycle arrest that prevents cancer and promotes biological ageing and inflammation. The molecular landscape of cellular senescence has been met with conflicting findings, likely due to contamination of senescent cells by neighbouring proliferating cells. Here, we developed a genetically encoded and tractable system to purify senescent cells in culture termed SenLect (Senescence seLection). SenLect is a genetic cassette that expresses mCherry-2A-PuroR under the control of the miR-146a senescence-activated promoter, which provides senescent cells with a selective advantage over proliferating cells in the presence of the antibiotic puromycin. We validated this one-step system in primary HUVECs and HeLa cervical cancer cells to purify live senescent cells following DNA damage (UV irradiation, hydrogen peroxide, and doxorubicin) and cell-cycle inhibition (palbociclib). Puromycin-selected cells were enriched for various senescence-related phenotypes, including cell cycle arrest, increased cell size, and lysosomal {beta}-galactosidase activity. By using SenLect to remove proliferating cells, we uncovered how the proteome is rewired during senescence induced by palbociclib. This improved the detection of protein signatures linked to the senescence-associated secretory phenotype (SASP), and metabolic shifts from mitochondrial respiration to glycolysis, and from nucleotide synthesis to catabolism. It also revealed sub-proteome level rewiring of organelles such as mitochondria, lysosomes and the nucleolus, as well as protein cohorts like kinases and core-essential proteins. SenLect provides a consistent and reliable method for isolating senescent cells that is suitable for downstream applications or analyses.

cell biology↗

bioRxiv2024

Cell-type specific autophagy in human leukocytes

Autophagy is a naturally conserved mechanism crucial for degrading and recycling damaged organelles and proteins to support cell survival. This process slows biological ageing and age-related disease in preclinical models. However, there has been little translation of autophagy to the clinic, and we have identified a lack of measurement tools for physiological human autophagy as a barrier. To address this, we have previously developed a direct measurement tool for autophagy in pooled human peripheral blood mononuclear cells (PBMCs) in the context of whole blood. In order to better understand how autophagy behaves and changes in humans, we measured human autophagic flux using flow cytometry in 19 cell sub-populations in whole blood to retain physiological flux. Autophagic flux was different between different cell types, being highest in B lymphocytes and lowest in T lymphocytes and monocytes. Autophagic flux also varied with sex, being higher in monocytes in females compared with males. In keeping with previous observations in humans, autophagy also increased with ageing at sub-population levels. Importantly, we found that only monocytes - specifically, non-classical monocytes - displayed increased autophagic flux following amino acid withdrawal, underscoring the importance of population selection for measurement of autophagic flux during nutrient restriction studies in humans. Collectively, these data show PBMC population level analysis improves sensitivity of human autophagic flux measurement.

cell biology↗

bioRxiv2024

Autophagy across tissues of aging mice

Autophagy is a waste-disposal pathway that protects against age-related pathology. It is widely accepted that autophagy declines with age, yet the role that sex and diet-related obesity play during aging remain unknown. Here, we present the most comprehensive in vivo study of autophagic flux to date. We employed transgenic mice overexpressing tandem-florescent LC3B (RFP-GFP-LC3B) to measure autophagic flux in the blood (PBMCs), heart, and motor cortex neurons of aging mice that were fed regular chow or a high-fat diet for 6-, 12- or 18-months. In male mice, aging decreased autophagic flux in the heart, increased it in the blood, and had no effect in motor cortex neurons. Age-dependent changes autophagic flux were less pronounced in female mice. High-fat diet influenced autophagic flux in the blood and heart of male but not female mice. Overall, we uncovered sexual dimorphisms that underpin how autophagy changes with age across different tissues and in response to a high-fat diet.

cell biology↗