The necroptotic protein MLKL accumulates in neurons over the lifespan, and their downregulation has the potential to improve cognition through reduced neuroinflammation and changes in the abundance of synaptic proteins and enzymes in brain tissue. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are still lacking. Here, we test whether the overexpression of MLKL in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial calcium levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP), as well as the ratio of oxidized/reduced glutathione as a proxy for oxidative stress. We found that MLKL overexpression leads to reduced cytosolic and mitochondrial Ca2+ elevations in response to selective cell permeabilization to this ion. On the contrary, Ca2+ signals were elevated after mobilizing Ca2+ from the endoplasmic reticulum. Irrespective of the stimulation paradigm, Neuro-2a cells presented higher lactate levels and lower glucose concentrations when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24-48h. At the proteomic level, we found evidence for decreased antioxidant defenses related to protein carbonylation, and increased abundance of molecular chaperones. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression contributes to both Ca2+ dyshomeostasis and metabolic reprogramming, which are cellular hallmarks of aging.

This entry contains the Thermo Scientific .raw files for targeted quantitative proteomics experiments carried out on at QEx Plus orbitrap system in the data independent acquisition mode. If you have any questions about this experiment, please contact Mike Kinter in the Aging and Metabolism Research Program at the Oklahoma Medical Research Foundation.