Cytokine-induced metabolic changes in neurons and astrocytes

The up-regulated AD-specific cytokine profile of IFNγ, IP-10/CXCL10, and IL-9, as determined by the multivariate modeling, was applied to primary neuron and astrocyte cultures derived from CD1 P0 neonates. The combination cytokine treatment was applied 72 h prior to experimentation in levels proportional to the concentrations we measured in 5xFAD 180-day hippocampus samples. Concentrations were centered in the nanomolar range previously used to study acute cytokine responses in neuron cultures [27 (link), 99 (link)]. Recombinant murine cytokines were purchased from Peprotech, IFN-γ (cat 315-05), IP-10/CXCL10 (cat 250-16), and IL-9 (cat 219-19), and reconstituted to 1 mg/mL in sterile water and diluted to 250 μg/mL (IL-9) or 10 μg/mL (IFNγ and IP-10/CXCL10) in 0.1% cell-culture grade bovine serum albumin (Sigma A9418) in 1X PBS. 5 nM IFNγ, 12 nM IP-10/CXCL10, and 500 nM IL-9 or an equal amount of 0.1% bovine serum albumin vehicle were added to the appropriate neuronal or glial cell culture medium for treatment. Neuron cultures were treated 9 or 10 days after plating, and astrocytes were treated, following the removal of non-adherent cells, at 50% confluency. After a 72-h stimulation, cells were assayed on the Seahorse XFe24 Extracellular Flux Analyzer or lysed for RNA isolation.