Creation and Characterization of Mitochondrial DNA‐Depleted Cell Lines with “Neuronal‐Like” Properties

Abstract

Mitochondrial dysfunction and attendant bioenergetic defects are increasingly recognized as playing an important role in neurodegenerative disorders. The increased attention on mitochondrial involvement points to the need for developing cell lines that have neuron-like characteristics for the genetic analysis and modeling of these diseases. We describe the creation of respiratorydeficient SH-SY5Y neuroblastoma cell lines (p°6415)by selectively depleting mitochondrial DNA through prolonged exposure to ethidium bromide. Oxygen consumption in these cells and activities of the electron transport chain enzyme complexes I and IV that contain subunits encoded by the mitochondrial genome are eliminated. In contrast, the function of complex II, a nuclear-encoded electron transport chain component, is largely intact in these cells. The p°64/5cells retain the ability to differentiate into cells with neuron-like phenotypes following treatment with phorbol ester or retinoic acid. Normal respiratory function is recovered by repopulation of p°64/5 cells with exogenous human platelet mitochondria. The p°6415cell line serves as a valuable model for the study of neurologic diseases suspected of involving mitochondrial dysfunction. Key Words: Mitochondria-Cytoplasmic hybrid-p cell-SH-SY5Y cells-Electron transport chain-Reactive oxygen species.

Mitochondrial function in Parkinson’s disease cybrids containing an nt2 neuron-like nuclear background

“…Platelet mitochondria from either PD or CT subjects were used to repopulate NT2 rho0 cells with mtDNA as previously described Binder et al, 2005). Untransformed cells were removed by withdrawal of pyruvate and uridine from the culture medium and substitution of dialyzed, heat inactivated fetal calf serum for non-dialyzed, heat inactivated fetal calf serum (Swerdlow et al, 1996;Miller et al, 1996). Specifically, the selection medium consisted of Optimen (Gibco Life Technologies) supplemented with 10% dialyzed, heat inactivated fetal calf serum, penicillin (50 U/mL), and streptomycin (50 lg/ mL).…”

Pluripotent stem cells and mitochondrial dysfunction

“…Moreover, in ρ˚ cells, a greater amount of mass is associated with lysosome and peroxidation production [65][66][67]. Remarkably, the differentiation of SH-SY5Y neuroblastoma cells into neuron-like cells is not affected by defective mitochondria, as indicated by the presence of long neurites and secretory granules, which are typical of differentiating neuroblastoma cells [68]. Therefore, targeting mitochondria metabolism or mutations in mitochondrial-related genes for inherited disease by using pluripotent stem cells is still a major therapeutic direction for cell therapy.…”

Redox Regulation of Store-Operated Ca2+Entry

“…Most cells have low but significant non-mitochondrial oxygen consumption (23). This has been estimated to be between 10%-20%, usually either by measuring oxygen consumption rates in the presence of rotenone or other ETC inhibitors, or by generating cell lines lacking mitochondrial DNA, the so-called ''rho 0'' cells (122,129). However, it remains unclear how much of the remaining oxygen consumption is utilized in the ER versus cytosol or within other organelles, and more importantly whether inhibiting mitochondrial function affects ER oxygen consumption.…”