AbstractBackgroundAstrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes involved in synaptic dysfunction during Alzheimer´s disease (AD). AD patients have imbalanced cholesterol metabolism shown by high levels of a side‐chain oxidized cholesterol known as 27‐hydroxycholesterol (27‐OH). Evidence from our laboratory has shown that elevated 27‐OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear.MethodOur results suggest that elevated 27‐OH decrease the astrocyte function in vivo in one model of brain cholesterol imbalance. To study the relationship between astrocytes and neurons we have developed a 3D co‐culture system (3D‐NAMs) that allows all of the cell types from mice embryos differentiate together in vitro.ResultWe have validated our 3D‐NAMs and show that it reproduces effects of 27‐OH observed in 2D and in vitro. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures but are present in 27‐OH overproducing mice. We uncover a mechanism of GLT‐1 dysfunction that leads to astrocyte activation, neuronal hyperexcitability and synaptic dysfunction based on the effects of 27‐OH on astrocytes.ConclusionWe propose 3D‐NAMs as tools that can give information about mechanisms of disease and will help elucidate the effectiveness and safety of drug interventions and report a new simple mechanism based on GLT‐1 downregulation in the presence of elevated 27‐OH as the culprit of hyperexcitability in neurons under hypercholesterolemia metabolic conditions.