Genotoxic and metabolic stress drive divergent senescence programs in human microglia

Microglial dysfunction is a hallmark of brain ageing linked to the accumulation of senescent microglial phenotypes that promote chronic neuroinflammation. Both genotoxic and metabolic stress have been implicated in microglial senescence; yet, whether distinct stressors shape senescence programs remain unclear. Here, we investigated the impact of chronic genotoxic and metabolic stress on senescence-associated phenotypes in the human microglia cell line HMC3. Cells were exposed to doxorubicin to induce sustained DNA damage or to chronic high-glucose conditions to model metabolic stress. Both stress paradigms induced characteristic senescence features including cellular and nuclear hypertrophy, increased senescence-associated {beta} galactosidase activity and reduced metabolic viability without significant cell loss. Both conditions activated the p53-p21 pathway and sustained DNA damage signalling, whereas metabolic stress additionally induced p16 expression and peripheral nuclear localisation of p21, suggesting divergence in senescence regulatory pathways. Mitochondrial alterations were evident under both conditions, Dox-induced stress was associated with downregulation of NRF2-TFAM signalling, whereas HG-induced stress induced NRF2-TFAM activation alongside increased KEAP1 expression, suggesting a constrained antioxidant response. This was accompanied by activation of mitochondrial and antioxidant stress responses that did not restore mitochondrial content. Furthermore, both stressors induced robust inflammatory activation, with genotoxic stress promoting a chemokine-rich senescence-associated secretory phenotype, while metabolic stress induced an interferon-associated inflammatory signature. Collectively, these findings demonstrated that chronic genotoxic and metabolic stress drive distinct yet overlapping senescence programs characterised by morphological changes, mitochondrial remodelling and persistent inflammatory activation. These stress-specific responses may differentially contribute to neurodegenerative processes and disease susceptibility.