ABSTRACT
Defective complex I (CI) is the most common type of oxidative phosphorylation disease, with an incidence of 1 in 5000 live births. Here, whole genome expression profiling of fibroblasts from CI deficient patients was performed to gain insight into the cell pathological mechanism. Our results suggest that patient fibroblasts responded to oxidative stress by Nrf2-mediated induction of the glutathione antioxidant system and Gadd45-mediated activation of the DNA damage response pathway. Furthermore, the observed reduced expression of selenoproteins, might explain the disturbed calcium homeostasis previously described for the patient fibroblasts and might be linked to endoplasmic reticulum stress. These results suggest that both glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency.
Subject(s)
Calcium/metabolism , Electron Transport Complex I/deficiency , Electron Transport Complex I/genetics , Metabolic Networks and Pathways/genetics , Mitochondrial Diseases/genetics , NF-E2-Related Factor 2/metabolism , Antioxidants/metabolism , Cell Cycle Proteins/metabolism , Child, Preschool , DNA Damage , Endoplasmic Reticulum Stress , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Glutathione/metabolism , Homeostasis/genetics , Humans , Infant , Infant, Newborn , Male , Mitochondrial Diseases/metabolism , Nuclear Proteins/metabolism , Oxidative Phosphorylation , Oxidative Stress , Selenoproteins/metabolismABSTRACT
Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.
Subject(s)
Antioxidants/therapeutic use , Fibroblasts/metabolism , Mitochondrial Diseases/drug therapy , Oxidative Phosphorylation , Oxidative Stress , Adolescent , Adult , Cell Line , Child , Child, Preschool , DNA Polymerase gamma , DNA, Mitochondrial/genetics , DNA-Directed DNA Polymerase/genetics , Female , Humans , Infant , Male , Mitochondrial Diseases/metabolism , Mutation , RNA, Transfer, Leu/genetics , Reactive Oxygen Species/metabolismSubject(s)
Ouabain/pharmacokinetics , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes , Ouabain/pharmacology , Protein Structure, Secondary , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Xenopus laevisABSTRACT
TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually induces a biphasic response consisting of a transitory increase in secretion (due to IP(3) mobilization of Ca(2+) from intracellular stores), followed by a sustained plateau phase of stimulated secretion (by protein kinase C-dependent influx of extracellular Ca(2+) through voltage-operated Ca(2+) channels). The melanotrope cell of the amphibian Xenopus laevis displays a unique secretory response to TRH, namely a broad transient but no sustained second phase, consistent with the observation that TRH induces a single Ca(2+) transient rather than the classic biphasic increase in [Ca(2+)](i). The purpose of the present study was to determine the signal transduction mechanism utilized by TRH in generating this Ca(2+) signaling response. Our hypothesis was that the transient reflects the operation of only one of the two signaling arms of the lipase (i.e., either IP(3)-induced mobilization of internal Ca(2+) or PKC-dependent influx of external Ca(2+)). Using video-imaging microscopy it is shown that the TRH-induced Ca(2+) transient is dramatically attenuated under Ca(2+)-free conditions and that thapsigargin has no noticeable effect on the TRH-induced transient. These observations indicate that an IP(3)-dependent mechanism plays no important role in the action of TRH. PKC also does not seem to be involved because an activator of PKC did not induce a Ca(2+) transient and an inhibitor of PKC did not affect the TRH response. Experiments with a bis-oxonol membrane potential probe showed that the TRH response also does not underlie a PKC-independent mechanism that would induce membrane depolarization. We conclude that the action of TRH on the Xenopus melanotrope does not rely on the classical phospholipase C-dependent mechanism.