Aging of the blood-brain barrier: Metabolic signatures of mouse brain endothelial cell senescence

The blood-brain barrier (BBB) serves as a critical boundary between the peripheral circulation and the brain. Brain endothelial cells (BECs), which form the inner lining of cerebral blood vessels, play a key role in maintaining this barrier. They express tight junction proteins that prevent molecules from leaking between cells into the brain and efflux transporters that actively remove substances from the brain. As we age, the BBB becomes leaky and dysfunctional, leading to cerebrovascular diseases and dementia. Here, we investigated how BECs become senescent, a major feature of aging, and change their metabolic profile. First, we demonstrate that both the chemotherapeutic doxorubicin, a known inducer of cellular senescence, as well as hydrogen peroxide, a mediator of oxidative stress, induce BEC senescence. This was indicated by positive senescence-associated β-galactosidase staining (P = 0.002 and P = 0.001 for doxorubicin and H₂O₂, respectively) and increased P16 gene expression (both P = 0.004). Next, we analyzed the metabolic profile of the senescent cells using mass spectrometry. We identified 47 significantly altered metabolites (P < 0.05) in cells and 65 in the supernatant samples of doxorubicin-induced senescent cells, as well as, 53 significantly altered metabolites in cells and 82 in the supernatant samples of H₂O₂-induced senescent cells. There was an overlap of 10 and 44 metabolites between both treatments in the cell and supernatant samples, respectively. Of those, 9 cellular and 12 supernatant metabolites have already been reported in the literature to be changed during senescence in the brain or other organs. We also identified new metabolites and provide pathway analyses of the significantly altered metabolites. These analyses are the first to characterize BBB cell senescence at a metabolite level and provide the basis for the development of novel therapeutic interventions for age-related neurological disorders.