High‐Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA‐Encoded Protein Levels in Eukaryotic Cells

Abstract

Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.

High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons

“…Hyperpolarization of DJ m is associated with higher energy demand, as found in differentiating neuroblasts (Voccoli and Colombaioni, 2009), dividing versus quiescent cells (Huang, 2002), and developmentally active cells (Daniele et al, 2017;Medina et al, 2002). Depolarization of DJ m is associated with diseased cells, aging, apoptosis, and toxic insults (Lezi and Swerdlow, 2012;Nadanaciva et al, 2011;Nicholls, 2004;Wagner et al, 2008). We measured DJ m and then ATP production in cultured neurons to identify chemicals that increase the functional output of mitochondria.…”

Quantitative imaging of tissue sections using infrared scanning technology

“…; Nadanaciva et al. ; Kumar et al. ), and here it was suggested that it has further merit as a reliable application in whole‐tissue section histological processing and quantification.…”

Gene Therapeutic Approaches for the Treatment of Mitochondrial Dysfunction in Parkinson’s Disease

“…Most likely, high energy-demanding tissues (such as the neurons of the CNS) require lower thresholds of mutated mtDNA to result in bioenergetic depletion. Depletion of mtDNA can also cause disrupted mtDNA protein synthesis and thus leads to insufficient energy production in the affected cells [34]. Furthermore, nDNA variation could be mainly responsible for these mtDNA changes, given the importance of nDNAencoded genes in mtDNA-related processes [35].…”