The assessment of mitochondrial respiratory chain (RCRC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RCRC enzymatic function (complexes I-IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.
Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein-a mutated bacterial dehalogenase which is fused to the protein of interest-and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of preexisting peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.
Highlights ► We showed that neuroglobin (Ngb) is localized in mitochondria in primary neurons. ► Ngb’s mitochondrial localization is increased after OGD. ► Ngb neuroprotection is correlated with increased mitochondrial distribution of Ngb. ► Mitochondria permeability transition pore is involved in Ngb’s mito translocation.
Maturation of neuronal synapses is thought to involve mitochondria. Bcl-x L protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-x L is abundant. Here, we report that overexpression of Bcl-x L postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-x L , overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-x L or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-x L appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-x L coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-x L protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-x L positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation. Bcl-2 synaptic transmission mitochondria cell death ABT-737
Background: Nanoparticulate titanium dioxide (nano-TiO2) enters the body through various routes and causes organ damage. Exposure to nano-TiO(2 )is reported to cause testicular injury in mice or rats and decrease testosterone synthesis, sperm number, and motility. Importantly, nano-TiO(2 )suppresses testosterone production by Leydig cells (LCs) and impairs the reproductive capacity of animals. Methods: In an attempt to establish the molecular mechanisms underlying the inhibitory effect of nano-TiO2 on testosterone synthesis, primary cultured rat LCs were exposed to varying concentrations of nano-TiO2 (0, 10, 20, and 40 mu g/mL) for 24 hours, and alterations in cell viability, cell injury, testosterone production, testosterone-related factors (StAR, 3 beta HSD, P450scc, SR-BI, and DAX1), and signaling molecules (ERK1/2, PKA, and PKC) were investigated. Results: The data show that nano-TiO2 crosses the membrane into the cytoplasm or nucleus, triggering cellular vacuolization and nuclear condensation. LC viability decreased in a time-dependent manner at the same nano-TiO2 concentration, nano-TiO(2 )treatment (10, 20, and 40 mu g/mL) decreased MMP (36.13%, 45.26%, and 79.63%), testosterone levels (11.40% and 44.93%), StAR (14.7%, 44.11%, and 72.05%), 3 beta HSD (26.56%, 50%, and 79.69%), pERK1/2 (27.83%, 63.61%, and 78.89%), PKA (47.26%, 70.54%, and 85.61%), PKC (30%, 50%, and 71%), SR-BI (16.41%, 41.79%, and 67.16%), and P450scc (39.41%, 55.26%, and 86.84%), and upregulated DAX1 (1.31-, 1.63-, and 3.18-fold) in primary cultured rat LCs. Conclusion: Our collective findings indicated that nano-TiO2-mediated suppression of testosterone in LCs was associated with regulation of ERK1/2-PKA-PKC signaling pathways.
Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide‐p‐trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that ‘mild uncoupling’ could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Δψ m ) were determined in parallel as a function of FCCP concentration. Δψ m dropped by less than 10 mV before respiratory control was lost. Conditions for the valid estimation of matrix superoxide levels were determined from the rate of oxidation of the matrix‐targeted fluorescent probe MitoSOX. No significant change in the level of matrix superoxide could be detected on addition of FCCP while respiratory control was retained, although cytoplasmic superoxide levels measured by dihydroethidium oxidation increased. ‘Mild uncoupling’ by 30 nmol/L FCCP did not alleviate neuronal dysregulation induced by glutathione depletion and significantly enhanced that due to menadione‐induced oxidative stress. Low protonophore concentrations enhanced N ‐methyl‐ d ‐aspartate receptor‐induced delayed calcium deregulation consistent with a decrease in the spare respiratory capacity available to match the bioenergetic demand of chronic receptor activation. It is concluded that the ‘mild uncoupling’ hypothesis is not supported by this model.
Implications of environmental toxins on the regulation of neutrophil function are being significantly appraised. Such effects can be varied and markedly different depending on the type and extent of chemical exposure, which results in direct damage to the immune system. Isocyanates with functional group (-NCO), are considered as highly reactive molecules with diverse industrial applications. However, patho-physiological implications resulting from their occupational and accidental exposures have not been well delineated. The present study was carried out to assess the immunotoxic response of isocyanates and their mode of action at a molecular level on cultured human neutrophils isolated from healthy human volunteers. Studies were conducted to evaluate both dose- and time-dependent (n = 3) response using N-succinimidyl N-methylcarbamate, a chemical entity that mimics the effects of methyl isocyanate in vitro. Measure of apoptosis through annexin-V-FITC/PI assay, active caspase-3, apoptotic DNA ladder assay and mitochondrial depolarization; induction of oxidative stress by CM-H(2)DCFDA and formation of 8'-hydroxy-2'-deoxyguanosine; and levels of antioxidant defense system enzyme glutathione reductase, multiplex cytometric bead array analysis to quantify the secreted cytokine levels (interleukin-8, interleukin-1beta, interleukin-6, interleukin-10, interferon-gamma, tumor necrosis factor, and interleukin-12p70) parameters were evaluated. Our results demonstrate that isocyanates induce neutrophil apoptosis via activation of mitochondrial-mediated pathway along with reactive oxygen species production; depletion in antioxidant defense states; and elevated pro-inflammatory cytokine response.
Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.