PROFILING ASTROCYTE DERIVED EXTRACELLULAR VESICLE IN RESPONSE TO SENESCENCE REVEALS BIOMARKER CANDIDATES FOR NEURODEGENERATION

Astrocytic senescence is increasingly recognized as a contributor to neuroinflammation. In addition to their diverse and well-characterised functions within the central nervous system, astrocytes actively mediate intercellular communication via extracellular vesicles (EV). EV are membrane-enclosed nanoparticles released by virtually all cell types into the extracellular space, where they facilitate intercellular communication by transferring bioactive cargo such as proteins, lipids, and nucleic acids. We investigated the effects of senescence on astrocyte-derived EV (AdEV) to identify biomarkers associated with early neurodegeneration.

Senescence was induced in H4 neuroglioma cells, a widely used astrocyte-like model, through two independent methods: pharmacological treatment with the CDK4/6 inhibitor Palbociclib and ionizing radiation to trigger the DNA damage response pathway. EV were isolated and purified from conditioned medium of control and senescent cultures using size exclusion chromatography and subsequently characterized by nanoflow cytometry.

Senescence induction in H4 cells was confirmed by abnormal nuclear morphology, Lamin B1 loss, and positive staining for senescence-associated beta galactosidase. Senescent AdEV did not show any difference in overall size and expression of CD9, CD63 and CD81, compared to their younger counterparts. Notably, our results showed a twofold increase in GLAST levels in senescent AdEV, suggesting that astrocyte senescence alters glutamate handling and extracellular glutamate regulation.

These findings demonstrate that senescence induces specific and measurable AdEV changes, highlighting the potential of AdEV as non-invasive biomarkers for early detection of age-dependent neurodegeneration.