Aging Human Endothelial Cells Affect Neuronal Viability and Function

Abstract

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer's disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for neurodegenerative disease through increased inflammation and cellular dysfunction. Despite the fact that aging is a key risk factor in neurodegenerative disease, preclinical studies frequently model age-related diseases using young rodents. In this study we used replicative senescence, a standard model of cell senescence that permits the study of cells as they progress to senescence to examine the differential effects of senescent vs. non-senescent primary human endothelial cells on a human neuronal cell line. We hypothesize that senescent (Sen) endothelial cells (ECs), which accumulate with aging, create a pro-inflammatory environment that adversely affects viability and function of neurons. First, we validated the replicative senescence process in human-derived ECs by quantifying β-galactosidase activity, a marker for cellular senescence, and the protein expression of Lamin B1, which decreases with senescence. After determining senescence phenotypes by passage number, ECs at varying stages of cellular senescence (EP: early passage, ES: early senescence, LS: late senescence) were co-cultured with LUHMES (Lund Human Mesencephalic cell line - dopamine-like neurons) to assess neuronal cell viability by measuring release of lactate dehydrogenase (LDH) into the culture medium and neurite outgrowth. Nitric oxide and extracellular vesicles released into the culture medium were also evaluated as indicators of an inflammatory environment. Co-culturing ECs with LUHMES decreased LUHMES cell viability as evidenced by an increase in LDH in co-cultures of LUHMES LS EC and by a decreased number of nuclei in LUHMES co-cultured with any of the EC types (EP, ES, LS). Co-culture with any of the EC types increased the area labeled by MAP2 and phospho-Neurofilament in LUHMES at 24h, indicating increased neurite outgrowth. At 48h, increased neurite outgrowth was observed in LUHMES+EP ECs and LUHMES+ES ECs co-cultures but not in the LUHMES + LP ECs. Nitric oxide was not increased by co-culture of LUHMES with any of the EC subtypes. The amount of extracellular vesicles as quantified by measuring the activity of acetylcholinesterase (AChE) was increased in co-cultures compared to LUHMES controls, but did not significantly differ from EC controls. In summary, ES ECs promote neurite outgrowth whereas LS ECs promote neurodegeneration. These findings suggest the importance of EC aging on neuronal cell morphology and viability, both parameters affected in age-related dementia, including Alzheimer's disease.