Characterizing the Electron Transport Chain: Functional Approach Using Extracellular Flux Analyzer on Mouse Tissue Samples.

The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O2 consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.

Fetal programming of polycystic ovary syndrome: Effects of androgen exposure on prenatal ovarian development.

Polycystic ovary syndrome (PCOS) is a common form of anovulatory infertility with a strong hereditary component but no candidate genes have been found. The inheritance pattern may be due to in utero androgen programming on gene expression and mitochondria. Mitochondria are maternally inherited and alterations to mitochondria after fetal androgen exposure may explain one of the mechanisms of fetal programming in PCOS. Our aim was to investigate the role of excessive prenatal androgens in ovarian development by identifying how hyperandrogenemia affects gene expression and mitochondria in neonatal ovary. Pregnant dams were injected with dihydrotestosterone on days 16-18 of pregnancy. Day 0 ovaries were collected for gene expression and mitochondrial studies. RNAseq showed differential gene expressions which were related to mitochondrial dysfunction, fetal gonadal development, oocyte maturation, metabolism, angiogenesis, and PCOS. Top 20 up and downregulated genes were validated with qPCR and Western Blot. Transcriptional pathways involved in folliculogenesis and genes involved in ovarian and mitochondrial function were dysregulated. Further, DHT exposure altered mitochondrial ultrastructure and function by increasing mitochondrial oxygen consumption and decreasing mitochondrial efficiency with increased proton leak within the first day of life. Our data indicates that one path that leads to PCOS begins at birth and is programmed in utero by androgens.

Role of the proteasome in protein oxidation and neural viability following low-level oxidative stress.

Numerous studies suggest that proteasome inhibition may play a causal role in mediating the increased levels of protein oxidation and neuron death observed in conditions associated with oxidative stress. In the present study we demonstrate that administration of non-toxic levels of oxidative stress does not result in impairment of 20S/26S proteasome activity, and actually increases the expression of specific proteasome subunits. Non-toxic levels of oxidative stress were observed to elevate the amount of protein oxidation in the presence of preserved proteasomal function, suggesting that proteasome inhibition may not mediate increases in protein oxidation following low-level oxidative stress. Preserving basal proteasome function appears to be critical to preventing the neurotoxicity of low-level oxidative stress, based on the ability of proteasome inhibitor treatment to exacerbate oxidative stress toxicity. Taken together, these data indicate that maintaining neural proteasome function may be critical to preventing neurotoxicity, but not the increase in protein oxidation, following low-level oxidative stress.

Polyglutamine expansion, protein aggregation, proteasome activity, and neural survival.

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.