Loss Of Mitochondrial Complex Research Articles

Western diet-induced obesity results in brain mitochondrial dysfunction in female Ossabaw swine.

Diet-induced obesity is implicated in the development of a variety of neurodegenerative disorders. Concurrently, the loss of mitochondrial Complex I protein or function is emerging as a key phenotype across an array of neurodegenerative disorders. Therefore, the objective of this study was to determine if Western diet (WD) feeding in swine [carbohydrate = 40.8% kCal (17.8% of total calories from high fructose corn syrup), protein = 16.2% kcal, fat = 42.9% kCal, and 2% cholesterol] would result in Complex I syndrome pathology. To characterize the effects of WD-induced obesity on brain mitochondria in swine, high resolution respirometry measurements from isolated brain mitochondria, oxidative phosphorylation Complex expression, and indices of oxidative stress and mitochondrial biogenesis were assessed in female Ossabaw swine fed a WD for 6-months. In line with Complex I syndrome, WD feeding severely reduced State 3 Complex I, State 3 Complex I and II, and uncoupled mitochondrial respiration in the hippocampus and prefrontal cortex (PFC). State 3 Complex I mitochondrial respiration in the PFC inversely correlated with serum total cholesterol. WD feeding also significantly reduced protein expression of oxidative phosphorylation Complexes I-V in the PFC. WD feeding significantly increased markers of antioxidant defense and mitochondrial biogenesis in the hippocampi and PFC. These data suggest WD-induced obesity may contribute to Complex I syndrome pathology by increasing oxidative stress, decreasing oxidative phosphorylation Complex protein expression, and reducing brain mitochondrial respiration. Furthermore, these findings provide mechanistic insight into the clinical link between obesity and mitochondrial Complex I related neurodegenerative disorders.

Fas-Activated Serine/Threonine Kinase Governs Cardiac Mitochondrial Complex I Functional Integrity in Ischemia/Reperfusion Heart

Cardiac energy homeostasis is strictly controlled by the mitochondrial complex-mediated respiration. In the heart, mitochondrial complex I is highly susceptible to functional and structural destroy after ischemia/reperfusion (I/R), thereby contributing to myocardial energy insufficiency and cardiomyocyte death. Fas-activated serine/threonine kinase (FASTK) is recently recognized as a key modulator of mitochondrial gene expression and respiration. However, the role of FASTK in cardiac I/R process is undetermined. Here, we show that FASTK expression was down-regulated in the post-I/R heart. The reactive oxygen species scavenger N-acetyl-L-cysteine reversed I/R-induced FASTK down-regulation. Genetic deletion of FASTK exacerbated I/R-induced cardiac dysfunction, enlarged myocardial infarct size, and increased cardiomyocyte apoptosis. Compared with the wild type control, the FASTK deficient heart exhibited a lower mRNA expression of NADH dehydrogenase subunit-6 (MTND6, a mitochondrial gene encoding a subunit of complex I) and was more vulnerable to I/R-associated complex I inactivation. Replenishment of FASTK expression via adenovirus-mediated gene delivery restored mitochondrial complex I activity and ameliorated cardiomyocyte death induced by I/R, whereas these beneficial effects were blocked by the co-treatment with rotenone, a specific complex I inhibitor. in vivo experiments further confirmed that cardiac overexpression of FASTK ameliorated I/R-related MTND6 down-regulation and mitochondrial complex I inactivation, thereby protecting the heart against I/R injury. Collectively, these data for the first time identify that the down-regulation of FASTK is a direct culprit behind the loss of mitochondrial complex I functional integrity and cardiac injury induced by I/R process. Targeting FASTK might be a promising and effective strategy for MI/R intervention.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumor growth1–6 and its inhibition has demonstrated efficacy in combination with immuno-, antiangiogenic, or oncogene targeted-therapies7–9. Furthermore, brain and lung human tumors display robust glucose oxidation by mitochondria10,11. However, it is not fully understood why a functional ETC is necessary for tumor growth in vivo. ETC function is coupled to the generation of ATP, i.e. oxidative phosphorylation (OXPHOS) and the production of TCA cycle metabolites. Mitochondrial complex I and II donate electrons to ubiquinone resulting in the generation of ubiquinol. This allows regeneration of NAD+ and FAD, necessary cofactors for oxidative TCA cycle flux. Mitochondrial complex III oxidizes ubiquinol back to ubiquinone to allow complexes I and II to function. The ubiquinone generated by complex III is also used as an electron acceptor by the only mitochondrial enzyme essential for de novo pyrimidine synthesis, dihydroorotate dehydrogenase (DHODH). We observed impaired tumor growth in cancer cells lacking mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX) 12 that also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II, or DHODH diminished the tumor growth of AOX expressing cancer cells deficient in mitochondrial complex III, highlighting the necessity of ubiquinone as an electron acceptor for tumor growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX) 13 targeted to the mitochondria or cytosol still failed to grow tumors. This suggests that NAD+ regeneration is not sufficient to drive tumor growth in vivo. Collectively, our findings indicate that tumor growth requires the ETC to oxidize ubiquinol which is essential to drive oxidative TCA cycle and DHODH activity.

Immune suppression with complex III

Loss of mitochondrial complex III in regulatory T cells reduces their suppressive activity without affecting their survival.

Prognostic significance of mitochondrial oxidative phosphorylation complexes: Therapeutic target in the treatment of retinoblastoma

PurposeAltered energy metabolism plays an important role in the development and progression of cancer. The objective of this study was to elucidate the role of mitochondrial oxidative phosphorylation complexes and their prognostic significance in retinoblastoma (Rb). MethodsImmunohistochemistry was performed on 109 primary enucleated retinoblastoma tissues for mitochondrial OXPHOS complexes and their expression was confirmed by western blotting. ResultsHistopathological high risk factors (HRFs) were identified in 42.2% cases. Mitochondrial OXPHOS complexes III, IV and V were expressed in more than 50% of primary retinoblastoma cases each whereas mitochondrial complex I was expressed in only 29/109 (26.60%) cases by immunohistochemistry. Loss of mitochondrial complex I correlated well with poor tumor differentiation and tumor invasion (p<0.05) whereas expression of mitochondrial complexes III, IV and V was associated with better survival (Kaplan–Meier method). ConclusionsThis was the first study predicting a relevant role of mitochondrial OXPHOS complexes and highlights the prognostic significance with patient outcome in retinoblastoma. Loss of mitochondrial complex I immunoexpression could prove to be a useful independent prognostic biomarker to identify high risk retinoblastoma patients. Differential expression of these mitochondrial complexes is a novel finding and may be used as an attractive future anticancer target in primary retinoblastoma tumors. Financial disclosureThe author(s) have no proprietary or commercial interest in any materials discussed in this article.

Maneb-induced dopaminergic neuronal death is not affected by loss of mitochondrial complex I activity: results from primary mesencephalic dopaminergic neurons cultured from individual Ndufs4+/+ and Ndufs4-/- mouse embryos.

Primary cultures from embryonic mouse ventral mesencephalon are widely used for investigating the mechanisms of dopaminergic neuronal death in Parkinson's disease models. Specifically, single mouse or embryo cultures from littermates can be very useful for comparative studies involving transgenic mice when the neuron cultures are to be prepared before genotyping. However, preparing single mouse embryo culture is technically challenging because of the small number of cells present in the mesencephalon of each embryo (150 000-300 000), of which only 0.5-5% are tyrosine hydroxylase-positive, dopaminergic neurons. In this study, we optimized the procedure for preparing primary mesencephalic neuron cultures from individual mouse embryos. Mesencephalic neurons were dissociated delicately, plated on Aclar film coverslips, and incubated in DMEM supplemented with fetal bovine serum for 5 days and then N2 supplement was added for 1 day, which resulted in the best survival of dopaminergic neurons from each embryo. Using this optimized method, we prepared mesencephalic neuron cultures from single Ndufs4 or Ndufs4 embryos and investigated the role of mitochondrial complex I in maneb-induced dopamine neuron death. Our results suggest that maneb toxicity to dopamine neurons is not affected by the loss of mitochondrial complex I activity in Ndufs4 cultures.

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson's disease model

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson's disease model

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson’s disease model

Mitochondrial complex I dysfunction is regarded as underlying dopamine neuron death in Parkinson's disease models. However, inactivation of the Ndufs4 gene, which compromises complex I activity, does not affect the survival of dopamine neurons in culture or in the substantia nigra pars compacta of 5-wk-old mice. Treatment with piericidin A, a complex I inhibitor, does not induce selective dopamine neuron death in either Ndufs4(+/+) or Ndufs4(-/-) mesencephalic cultures. In contrast, rotenone, another complex I inhibitor, causes selective toxicity to dopamine neurons, and Ndufs4 inactivation potentiates this toxicity. We identify microtubule depolymerization and the accumulation of cytosolic dopamine and reactive oxygen species as alternative mechanisms underlying rotenone-induced dopamine neuron death. Enhanced rotenone toxicity to dopamine neurons from Ndufs4 knockout mice may involve enhanced dopamine synthesis caused by the accumulation of nicotinamide adenine dinucleotide reduced. Our results suggest that the combination of disrupting microtubule dynamics and inhibiting complex I, either by mutations or exposure to toxicants, may be a risk factor for Parkinson's disease.

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: Implications in excitotoxicity

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by β- N-oxalyl amino- l-alanine ( l-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. l-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or l-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with l-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to l-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to l-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.

Parkinson's Disease Brain Mitochondrial Complex I Has Oxidatively Damaged Subunits and Is Functionally Impaired and Misassembled

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

Glutaredoxin is essential for maintenance of brain mitochondrial complex I: studies with MPTP.

Mitochondrial complex I dysfunction is implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Identification of factors involved in maintenance and restoration of complex I function could potentially help to develop prophylactic and therapeutic strategies for treatment of this class of disorders. Down-regulation of glutaredoxin (thioltransferase, a thiol disulfide oxido-reductase) using antisense oligonucleotides results in the loss of mitochondrial complex I activity in mouse brain. 1-Methyl-4-phenyl-1,2,3,6,tetrahydro-pyridine (MPTP), the neurotoxin that causes Parkinson's disease-like symptoms in primates and dopaminergic cell loss in mice, acts through the inhibition of complex I. Regeneration of complex I activity in the striatum occurs concurrently with increase in glutaredoxin activity, 4 h after the neurotoxic insult, and is mediated through activation of activating protein-1. Down-regulation of glutaredoxin using anti-sense oligonucleotides prevents recovery of complex I in the striatum after MPTP treatment, providing support for the critical role for glutaredoxin in recovery of mitochondrial function in brain. Maintenance and restoration of protein thiol homeostasis by glutaredoxin may be important factors in preventing complex I dysfunction.

Thiol Oxidation and Loss of Mitochondrial Complex I Precede Excitatory Amino Acid-Mediated Neurodegeneration

Human ingestion of "chickling peas" from the plant Lathyrus sativus, which contains an excitatory amino acid, L-BOAA (L-beta-N-oxalylamino-L-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to L-BOAA, but not its optical enantiomer D-BOAA, causes mitochondrial dysfunction as evidenced by loss of complex I activity in vitro in male mouse brain slices and in vivo in selected regions of mouse CNS (lumbosacral cord and motor cortex). Loss of complex I activity in lumbosacral cord after L-BOAA administration to mice was accompanied by concurrent loss of glutathione. The inhibited complex I activity in mitochondria isolated from lumbosacral cord of animals treated with L-BOAA rebounded after incubation with the thiol-reducing agent dithiothreitol, indicating that oxidation of protein thiols to disulfides was responsible for enzyme inhibition. The inhibition of complex I could be abolished by pretreatment with antioxidant thiols such as glutathione ester and alpha-lipoic acid. Chronic treatment of male mice, but not female mice, with L-BOAA resulted in loss of complex I activity and vacuolation and dendritic swelling of neurons in the motor cortex and lumbar cord, paralleling the regionality of the aforementioned biochemical effects on CNS mitochondria. These results support the view that thiol oxidation and concomitant mitochondrial dysfunction (also implicated in other neurodegenerative disorders), occurring downstream of glutamate receptor activation by L-BOAA, are primary events leading to neurodegeneration. Maintenance of protein thiol homeostasis by thiol delivery agents could potentially offer protection against excitotoxic insults such as those seen with L-BOAA.

Parkinson's disease: from etiology to treatment.

We review the recent progress in the research of the etiology, pathogenesis and treatment of Parkinson's disease. It has been postulated that mitochondrial respiratory failure and oxidative stress are two major contributors to nigral cell death in Parkinson's disease. Loss of mitochondrial complex I and the alpha-ketoglutarate dehydrogenase complex in the substantia nigra has been reported. Evidence to indicate oxidative stress includes a high dopamine content, increase in superoxide dismutase activities, increase in iron, and decrease in glutathione in the substantia nigra. The question posed is which one occurs first. We believe mitochondrial respiratory failure occurs first, because slowing down of the electron transport induces an increase in the formation of activated oxygen species. The primary cause of Parkinson's disease is still unknown, but we believe the interaction of environmental toxins and genetic predispositions is important. In this respect, molecular genetic studies on familial Parkinson's disease are very important.