Variable toxicological response to the loss of OXPHOS through 1-methyl-4-phenylpyridinium-induced mitochondrial damage and anoxia in diverse neural immortal cell lines

Abstract

Immortal cell lines are used to investigate various aspects of neurodegeneration. These cells display high glycolytic turnover rate and produce an abundant amounts of lactate. Our previous studies indicate that these cells survive the loss of mitochondrial oxidative phosphorylation (OXPHOS) with ample glucose supply. In the current study, we investigate if cell type (w/variation in basal metabolic rate (MR)), can alter glucose utilization patterns which in turn may affect LC₅₀ for the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP⁺) in various cell lines. The data obtained indicate that cell lines MRs examined were generally consistent with the average of species adult body weight where mouse N-2A > rat-PC-12 > human SH-SY5Y. A higher MR was associated with accelerated utilization of glucose and earlier cell death with MPP⁺: LC₅₀ mouse = 294 µM, rat = 695 µM, and human = 5.25 mM at 24 h. Cell death appears to be a function of the velocity by which glucose disappears, leading to the failure of glycolysis and subsequent halt of energy production. Similar effects were also observed at higher plating densities where the demand for glucose is amplified. A time-lapse study of MPP⁺ toxicity (0–36 h) in N-2A cells indicates that an anaerobic shift occurs as early as 2 h (evidenced by a rise in lactate), followed by a descent in glucose concentrations at 4 h and exhaustion of glucose supplies at 22 h which was associated with the first detectable sign of cell death. It was also noted that MPP⁺ toxicity was not associated with the generation of reactive oxygen species (O ⁻₂ , H₂0₂, and NO₂) and was not attenuated by adding catalase or superoxide dismutase to the media. On the other hand, MPP⁺ toxicity was reversed by providing additional supply of glucose, pyruvate ± mitochondrial monocarboxylate transporter blocker (α-cyano-4-HCA), or pyruvate ± pyruvate dehydrogenase inhibitor (octanoyl-CoA), suggesting that the exclusive anaerobic survival compensates for the loss of OXPHOS by MPP⁺. To examine if neuroblastoma were capable of surviving the deprivation of O₂ for 24 h, a range of hypoxia to anoxia was established with various concentrations of dithionite. The data suggest that cell lines examined continue to thrive when incubated with high-glucose media (25 mM). In summary, vulnerability of immortal neuroblastoma cell lines to MPP⁺ toxicity is dependent upon glucose concentrations within the media and cell MR, which indirectly dominates the velocity of glucose use and its end point disappearance, leading to cell death by ergogenic failure.