Neuronal and Astroglial Cells Are Characterized by Unique Redox and Metabolic Features

Abstract

ObjectiveTo understand cell‐specific differences in energy generation, as well as methionine‐and antioxidant‐pathways in SH‐SY5Y neuroblastoma and U87MG glioblastoma cells.BackgroundDifferential metabolic activities in astrocytes versus neurons allow astrocytes to provide a supportive metabolic environment for neurons. Neurons have a higher metabolic rate than astrocytes to support ATP generation for maintaining their signaling activities. As oxidative phosphorylation generates reactive oxygen species, the rate of oxidative phosphorylation in a cell must be balanced by maintaining adequate antioxidant status. Cysteine is required to generate glutathione, which is one of the primary antioxidants in cells. Cysteine in the brain is predominantly found in its oxidized form, cystine, which neurons cannot import. Cysteine can be also be created from the transsulfuration pathway from cystathionine derived from homocysteine. However, it is thought that the transsulfuration pathway in neurons is partially blocked. Therefore, neurons must obtain cysteine by breaking down glutathione exported from astrocytes. SH‐SY5Y cells are neuron‐like cells and U87MG cells are astroglial‐like. Therefore, both cell lines are often used as experimental models of neurons and astrocytes, respectively. We examined metabolites in the methionine cycle and associated antioxidant‐generating pathways and bioenergetic status in both cell lines to see whether they recapitulated features of bona fide neurons and astrocytes.MethodsSH‐SY5Y neuroblastoma cells and U87MG glioblastoma cells were grown in T75 flasks in DMEM with 10% FBS and 1% penicillin‐streptomycin‐fungizone. Cells were plated in 6‐well plates. Thiol‐containing metabolites in methionine cycle and transsulfuration pathway were measured through LC/MS. Metabolite levels were normalized to cellular protein content with bicinchoninic acid (BCA) assay. Mitochondrial oxidation (Oxygen Consumption Rate, OCR) and glycolytic‐fermentation (Extracellular Acidification Rate, ECAR) activity were measured with a Seahorse XFp apparatus.ResultsMethionine was approximately 2.3 times higher in U87MG cells than SH‐SY5Y cells. S‐adenosylmethionine (SAM) and S‐adenosylhomocysteine (SAH) were comparable in both cell types. Cystathionine levels in SH‐SY5Y cells were 106 times those in U87MG cells. Lanthionine was 65% higher and cysteine was 47% higher in U87MG cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were not significantly different between SH‐SY5Y or U87MG cells. GSH/GSSG was over 4‐times higher in U87MG cells. Basal OCR and basal ECAR were higher in SH‐SY5Y cells, but OCR/ECAR was 71% higher in U87MG cells.ConclusionThe striking difference in cystathionine levels between SH‐SY5Y and U87MG cells support that there are differences in transsulfuration between neurons and astrocytes. The increased GSH/GSSG in U87MG cells also implies that astrocytes maintain antioxidant status differently and perhaps more efficiently than neurons. Higher basal OCR and ECAR rates in SH‐SY5Y cells corroborate the notion of increased energy need by neurons.Support or Funding InformationThis research was supported with a fellowship from the American Foundation for Pharmaceutical Education (AFPE).