Assessing Mitochondrial Function in In Vitro and Ex Vivo Models of Huntington’s Disease

Abstract

Mitochondrial dysfunction has gained a preponderant role in the pathogenesis of Huntington's disease (HD). Mutant huntingtin (mHTT) directly interacts with mitochondria in a deleterious manner. As the central hub of the cell, not only mitochondrial bioenergetics is affected but there is also diminished mitochondrial membrane potential (Δψ ) and altered production of reactive oxygen species (ROS). Restoration of mitochondrial function has proven to be a major player in the search and establishment of therapeutics for HD patients. As such, performing an overall study of mitochondrial function is crucial. In this chapter, we describe some methodologies used to study mitochondrial function by determining the oxygen consumption, changes in Δψ, mitochondrial calcium handling, and levels of mitochondrial ROS. Here we focus on biological samples derived from HD versus control cells and/or animal models, namely functional isolated brain mitochondria, an ex vivo animal model, and cultured cells, including cell lines and primary neural cultures, as in vitro models.

Mitochondrial dysfunction and oxidative stress contribute to cognitive and motor impairment in FOXP1 syndrome

“…Cytochrome c release was determined by calculating the ratio of occurrence in the mitochondria to cytoplasm. The isolation of mitochondrial and cytosolic proteins was conducted as previously described [22]. Cytochrome c, Cox IV (a marker for mitochondrial proteins) and Gapdh (a marker for cytosolic proteins) were evaluated by western blot.…”

Role of defective calcium regulation in cardiorespiratory dysfunction in Huntington’s disease

“…While the mechanisms involved in HD remain unclear, several hypotheses have been put forward: mutated huntingtin aggregates form inclusion bodies inside neurons, and insoluble huntingtin causes mitochondrial dysfunction, as well as Ca 2+ dyshomeostasis and defective protein-protein interactions and vesicular transport of proteins including neurotransmitter receptors, ultimately leading to neuronal death. Moreover, mutated huntingtin microaggregates increase ROS production in the brain (85). Here we show increased ROS production in Q175 brains and increased RyR2 oxidation, which causes ER Ca 2+ leak (Supplemental Figure 3A).…”

Microbial BMAA elicits mitochondrial dysfunction, innate immunity activation, and Alzheimer’s disease features in cortical neurons

“…Pre-frontal cortex tissue from C57BL/6 mice (adult females) was washed in ice-cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, and pH 7.2/KOH. Cortical mitochondria were then isolated using discontinuous Percoll density gradient centrifugation, according to Ferreira and collaborators with some minor modifications [ 35 ]. For this purpose, pre-frontal cortical tissues were homogenized with 25 up and down strokes in Dounce All-Glass Tissue Grinder (Kontes Glass Co., Vineland, NJ, USA) using pestle A (clearance, 0.07–0.12 mm) followed by 25 up and down strokes with pestle B (clearance, 0.02–0.056 mm).…”

“…Mitochondria (5 μg) were resuspended in washing buffer and loaded in each well. Isolated mitochondria were kept on ice until use on Seahorse apparatus in an assay based upon fluorimetric detection of O 2 and H + levels [ 35 , 36 ]. Isolated mitochondria were solubilized in ice-cold mitochondrial assay solution (MAS) containing 70 mM sucrose, 220 mM mannitol, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 2 mM HEPES, 1 mM EGTA, pH 7.2, plus 10 mM succinate, and 2 μM rotenone substrates.…”