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1.
Curr Opin Nephrol Hypertens ; 32(6): 528-536, 2023 11 01.
Article in English | MEDLINE | ID: mdl-37661939

ABSTRACT

PURPOSE OF REVIEW: MtDNA copy number (CN), a putative noninvasive biomarker of mitochondrial dysfunction, is associated with renal disease. The purpose of this review is to describe studies which measured human blood mtDNA-CN in the context of chronic kidney disease (CKD), and to evaluate its potential as a clinical biomarker of kidney disease. RECENT FINDINGS: Following on from small scale cross-sectional studies implicating mtDNA-CN changes in diabetic kidney disease, recent large scale population studies provide compelling evidence of the association of mtDNA-CN and risk of renal disease in the general population and poor outcomes in CKD patients. SUMMARY: The kidney has high bioenergetic needs, renal cells are rich in mitochondrial content containing 100s to 1000s of mtDNA molecular per cell. MtDNA has emerged as both a potential mediator, and a putative biomarker of renal disease. Damage to mtDNA can result in bioenergetic deficit, and reduced MtDNA levels in the blood have been shown to correlate with CKD. Furthermore, leakage of mtDNA outside of mitochondria into the cytosol/periphery can directly cause inflammation and is implicated in acute kidney injury (AKI). Recent large-scale population studies show the association of mtDNA-CN and renal disease and provide a strong basis for the future evaluation of circulating DNA-CN in longitudinal studies to determine its utility as a clinical biomarker for monitoring renal function.


Subject(s)
DNA, Mitochondrial , Renal Insufficiency, Chronic , Humans , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Cross-Sectional Studies , Mitochondria , Kidney/metabolism , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/genetics , Biomarkers/metabolism
2.
EMBO J ; 42(7): e108533, 2023 04 03.
Article in English | MEDLINE | ID: mdl-36825437

ABSTRACT

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.


Subject(s)
Mitochondria , Proteins , Humans , HeLa Cells , Macromolecular Substances/metabolism , Proteins/metabolism , Solvents/metabolism , Mitochondria/metabolism
3.
PLoS One ; 17(1): e0262544, 2022.
Article in English | MEDLINE | ID: mdl-35015774

ABSTRACT

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious dose-limiting side effect of several first-line chemotherapeutic agents including paclitaxel, oxaliplatin and bortezomib, for which no predictive marker is currently available. We have previously shown that mitochondrial dysfunction is associated with the development and maintenance of CIPN. The aim of this study was to evaluate the potential use of mitochondrial DNA (mtDNA) levels and complex I enzyme activity as blood biomarkers for CIPN. Real-time qPCR was used to measure mtDNA levels in whole blood collected from chemotherapy- and vehicle-treated rats at three key time-points of pain-like behaviour: prior to pain development, at the peak of mechanical hypersensitivity and at resolution of pain-like behaviour. Systemic oxaliplatin significantly increased mtDNA levels in whole blood prior to pain development. Furthermore, paclitaxel- and bortezomib-treated animals displayed significantly higher levels of mtDNA at the peak of mechanical hypersensitivity. Mitochondrial complex I activity in whole blood was assessed with an ELISA-based Complex I Enzyme Activity Dipstick Assay. Complex I activity was not altered by any of the three chemotherapeutic agents, either prior to or during pain-like behaviour. These data demonstrate that blood levels of mtDNA are altered after systemic administration of chemotherapy. Oxaliplatin, in particular, is associated with higher mtDNA levels before animals show any pain-like behaviour, thus suggesting a potential role for circulating mtDNA levels as non-invasive predictive biomarker for CIPN.


Subject(s)
Antineoplastic Agents/toxicity , Biomarkers/blood , DNA, Mitochondrial/blood , DNA, Mitochondrial/genetics , Mitochondria/pathology , Peripheral Nervous System Diseases/diagnosis , Animals , Male , Mitochondria/drug effects , Mitochondria/genetics , Peripheral Nervous System Diseases/blood , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/genetics , Rats , Rats, Sprague-Dawley
4.
Eur J Clin Invest ; 52(3): e13622, 2022 Mar.
Article in English | MEDLINE | ID: mdl-34050922

ABSTRACT

According to the 'multiple-hit' hypothesis, several factors can act simultaneously in nonalcoholic fatty liver disease (NAFLD) progression. Increased nitro-oxidative (nitroso-oxidative) stress may be considered one of the main contributors involved in the development and risk of NAFLD progression to nonalcoholic steatohepatitis (NASH) characterized by inflammation and fibrosis. Moreover, it has been repeatedly postulated that mitochondrial abnormalities are closely related to the development and progression of liver steatosis and NAFLD pathogenesis. However, it is difficult to determine with certainty whether mitochondrial dysfunction or oxidative stress are primary events or a simple consequence of NAFLD development. On the one hand, increasing lipid accumulation in hepatocytes could cause a wide range of effects from mild to severe mitochondrial damage with a negative impact on cell fate. This can start the cascade of events, including an increase of cellular reactive nitrogen species (RNS) and reactive oxygen species (ROS) production that promotes disease progression from simple steatosis to more severe NAFLD stages. On the other hand, progressing mitochondrial bioenergetic catastrophe and oxidative stress manifestation could be considered accompanying events in the vast spectrum of abnormalities observed during the transition from NAFL to NASH and cirrhosis. This review updates our current understanding of NAFLD pathogenesis and clarifies whether mitochondrial dysfunction and ROS/RNS are culprits or bystanders of NAFLD progression.


Subject(s)
Mitochondria/metabolism , Non-alcoholic Fatty Liver Disease/etiology , Oxidative Stress , Humans
5.
Methods Mol Biol ; 2277: 247-268, 2021.
Article in English | MEDLINE | ID: mdl-34080155

ABSTRACT

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per µL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.


Subject(s)
Cell-Free Nucleic Acids/blood , DNA, Mitochondrial/blood , Real-Time Polymerase Chain Reaction/methods , Cell-Free Nucleic Acids/isolation & purification , DNA, Mitochondrial/isolation & purification , Humans , Leukocytes, Mononuclear/physiology , Real-Time Polymerase Chain Reaction/instrumentation
6.
Int J Obes (Lond) ; 44(12): 2382-2393, 2020 12.
Article in English | MEDLINE | ID: mdl-33033395

ABSTRACT

OBJECTIVES: We hypothesised that maternal diet-induced-obesity has adverse consequences for offspring energy expenditure and susceptibility to obesity in adulthood, and that the prebiotic polydextrose (PDX) would prevent the consequences of programming by maternal obesity. METHODS: Female mice were fed a control (Con) or obesogenic diet (Ob) for 6 weeks prior to mating and throughout pregnancy and lactation. Half the obese dams were supplemented with 5% PDX (ObPDX) in drinking water throughout pregnancy and lactation. Offspring were weaned onto standard chow. At 3 and 6 months, offspring energy intake (EI) and energy expenditure (EE by indirect calorimetry) were measured, and a glucose-tolerance test performed. Offspring of control (OffCon), obese (OffOb) and PDX supplemented (OffObP) dams were subsequently challenged for 3 weeks with Ob, and energy balanced reassessed. Potential modifiers of offspring energy balance including gut microbiota and biomarkers of mitochondrial activity were also evaluated. RESULTS: Six-month-old male OffOb demonstrated increased bodyweight (BW, P < 0.001) and white adipose tissue mass (P < 0.05), decreased brown adipose tissue mass (BAT, P < 0.01), lower night-time EE (P < 0.001) versus OffCon, which were prevented in OffObP. Both male and female OffOb showed abnormal glucose-tolerance test (peak [Glucose] P < 0.001; AUC, P < 0.05) which was prevented by PDX. The Ob challenge resulted in greater BW gain in both male and female OffOb versus OffCon (P < 0.05), also associated with increased EI (P < 0.05) and reduced EE in females (P < 0.01). OffObP were protected from accelerated BW gain on the OB diet compared with controls, associated with increased night-time EE in both male (P < 0.05) and female OffObP (P < 0.001). PDX also prevented an increase in skeletal muscle mtDNA copy number in OffOb versus OffCon (P < 0.01) and increased the percentage of Bacteroides cells in faecal samples from male OffObP relative to controls. CONCLUSIONS: Maternal obesity adversely influences adult offspring energy balance and propensity for obesity, which is ameliorated by maternal PDX treatment with associated changes in gut microbiota composition and skeletal muscle mitochondrial function.


Subject(s)
Glucans/administration & dosage , Obesity, Maternal/complications , Prebiotics/administration & dosage , Prenatal Exposure Delayed Effects , Animals , Body Composition , Body Weight , Diet , Energy Intake , Energy Metabolism , Female , Gastrointestinal Microbiome , Glucose/metabolism , Glucose Tolerance Test , Homeostasis , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Pregnancy
7.
FASEB J ; 34(9): 12278-12288, 2020 09.
Article in English | MEDLINE | ID: mdl-32729179

ABSTRACT

Circulating mitochondrial DNA (mtDNA), widely studied as a disease biomarker, comprises of mtDNA located within mitochondria, indicative of mitochondrial function, and cell-free (cf) mtDNA linked to inflammation. The purpose of this study was to determine the ranges of, and relationship between, cellular and cf mtDNA in human blood. Whole blood from 23 controls (HC) and 20 patients with diabetes was separated into peripheral blood mononuclear cells (PBMCs), plasma, and serum. Total DNA was isolated and mtDNA copy numbers were determined using absolute quantification. Cellular mtDNA content in PBMCs was higher than in peripheral blood and a surprisingly high level of cf mtDNA was present in serum and plasma of HC, with no direct relationship between cellular and cf mtDNA content within individuals. Diabetes patients had similar levels of cellular mtDNA compared to healthy participants but a significantly higher cf mtDNA content. Furthermore, only in patients with diabetes, we observed a correlation between whole blood and plasma mtDNA levels, indicating that the relationship between cellular and cf mtDNA content is affected by disease status. In conclusion, when evaluating mtDNA in human blood as a biomarker of mitochondrial dysfunction, it is important to measure both cellular and cf mtDNA.


Subject(s)
Cell-Free Nucleic Acids/blood , DNA, Mitochondrial/blood , Adult , Biomarkers/blood , Diabetes Mellitus/blood , Diabetes Mellitus/physiopathology , Female , Humans , Male , Middle Aged , Mitochondria/physiology
8.
Stem Cells ; 38(4): 574-584, 2020 04.
Article in English | MEDLINE | ID: mdl-31912945

ABSTRACT

Pretransplant islet culture is associated with the loss of islet cell mass and insulin secretory function. Insulin secretion from islet ß-cells is primarily controlled by mitochondrial ATP generation in response to elevations in extracellular glucose. Coculture of islets with mesenchymal stromal cells (MSCs) improves islet insulin secretory function in vitro, which correlates with superior islet graft function in vivo. This study aimed to determine whether the improved islet function is associated with mitochondrial transfer from MSCs to cocultured islets. We have demonstrated mitochondrial transfer from human adipose MSCs to human islet ß-cells in coculture. Fluorescence imaging showed that mitochondrial transfer occurs, at least partially, through tunneling nanotube (TNT)-like structures. The extent of mitochondrial transfer to clinically relevant human islets was greater than that to experimental mouse islets. Human islets are subjected to more extreme cellular stressors than mouse islets, which may induce "danger signals" for MSCs, initiating the donation of MSC-derived mitochondria to human islet ß-cells. Our observations of increased MSC-mediated mitochondria transfer to hypoxia-exposed mouse islets are consistent with this and suggest that MSCs are most effective in supporting the secretory function of compromised ß-cells. Ensuring optimal MSC-derived mitochondria transfer in preculture and/or cotransplantation strategies could be used to maximize the therapeutic efficacy of MSCs, thus enabling the more widespread application of clinical islet transplantation.


Subject(s)
Diabetes Mellitus, Experimental/therapy , Insulin-Secreting Cells/metabolism , Islets of Langerhans Transplantation/methods , Mesenchymal Stem Cells/metabolism , Mitochondria/metabolism , Animals , Cells, Cultured , Humans , Mice
9.
Stem Cells ; 38(3): 369-381, 2020 03.
Article in English | MEDLINE | ID: mdl-31778245

ABSTRACT

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.


Subject(s)
Blastocyst/metabolism , Mitochondria/metabolism , Thyroid Hormones/metabolism , Female , Humans , Oxidative Phosphorylation , Pregnancy
10.
Int J Mol Sci ; 20(24)2019 Dec 11.
Article in English | MEDLINE | ID: mdl-31835862

ABSTRACT

Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm the presence of, and determine the location and prevalence of, mtDNA mutation in DR. DNA isolated from peripheral blood from diabetes patients (n = 59) with and without DR was used to amplify specific mtDNA regions which were digested with surveyor nuclease S1 to determine the presence and location of heteroplasmic mtDNA mutations were present. An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) of DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high-prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome supports the view that mtDNA damage contributes to DR. The exact location and functional impact of these mutations remains to be determined.


Subject(s)
Diabetic Retinopathy/genetics , Genome, Mitochondrial/genetics , Mutation/genetics , Adult , Aged , Aged, 80 and over , Cohort Studies , DNA Damage , DNA, Mitochondrial/genetics , Diabetic Retinopathy/pathology , Female , Humans , Male , Middle Aged , Pilot Projects
11.
Cells ; 8(10)2019 10 08.
Article in English | MEDLINE | ID: mdl-31597406

ABSTRACT

Non-alcoholic fatty liver disease (NAFLD), an increasingly prevalent and underdiagnosed disease, is postulated to be caused by hepatic fat mediated pathological mechanisms. Mitochondrial dysfunction is proposed to be involved, but it is not known whether this is a pathological driver or a consequence of NAFLD. We postulate that changes to liver mitochondrial DNA (mtDNA) are an early event that precedes mitochondrial dysfunction and irreversible liver damage. To test this hypothesis, we evaluated the impact of diet on liver steatosis, hepatic mtDNA content, and levels of key mitochondrial proteins. Liver tissues from C57BL/6 mice fed with high fat (HF) diet (HFD) and Western diet (WD, high fat and high sugar) for 16 weeks were used. Steatosis/fibrosis were assessed using haematoxylin and eosin (H&E) Oil Red and Masson's trichome staining and collagen content. Total DNA was isolated, and mtDNA content was determined by quantifying absolute mtDNA copy number/cell using quantitative PCR. Selected mitochondrial proteins were analysed from a proteomics screen. As expected, both HFD and WD resulted in steatosis. Mouse liver contained a high mtDNA content (3617 ± 233 copies per cell), which significantly increased in HFD diet, but this increase was not functional, as indicated by changes in mitochondrial proteins. In the WD fed mice, liver dysfunction was accelerated alongside downregulation of mitochondrial oxidative phosphorylation (OXPHOS) and mtDNA replication machinery as well as upregulation of mtDNA-induced inflammatory pathways. These results demonstrate that diet induced changes in liver mtDNA can occur in a relatively short time; whether these contribute directly or indirectly to subsequent mitochondrial dysfunction and the development of NAFLD remains to be determined. If this hypothesis can be substantiated, then strategies to prevent mtDNA damage in the liver may be needed to prevent development and progression of NAFLD.


Subject(s)
DNA Damage , DNA, Mitochondrial , Diet, High-Fat/adverse effects , Diet, Western/adverse effects , Mitochondrial Proteins/analysis , Non-alcoholic Fatty Liver Disease/physiopathology , Animals , Disease Models, Animal , Liver/metabolism , Liver/physiopathology , Male , Mice , Mice, Inbred C57BL , Mitochondria, Liver/metabolism , Mitochondria, Liver/physiology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/metabolism , Oxidative Phosphorylation , Proteome/analysis
12.
Sci Rep ; 9(1): 11386, 2019 08 06.
Article in English | MEDLINE | ID: mdl-31388037

ABSTRACT

Diabetes increases the risk of Alzheimer's disease (AD), and mitochondrial dysfunction is implicated in both diseases, however the impact of both diabetes and AD on brain mitochondria is not known. We measured mitochondrial DNA (mtDNA), an indicator of mitochondrial function, in frontal, parietal, and cerebellar regions of post-mortem human brains (n = 74) from non-cognitively impaired controls (NCI), mild-cognitively impaired (MCI) and AD cases. In a subset of parietal cortices, we measured mRNAs corresponding to cell types and mitochondrial function and semi-automated stereological assessment was performed on immune-staining of parietal cortex sections. mtDNA showed significant regional variation, highest in parietal cortex, and lowest in cerebellum. Irrespective of cognitive status, all brain regions had significantly higher mtDNA in diabetic cases. In the absence of diabetes, AD parietal cortices had decreased mtDNA, reduced MAP2 (neuronal) and increased GFAP (astrocyte) mRNA, relative to NCI. However, in the presence of diabetes, we did not observe these AD-related changes, suggesting that the pathology observed in diabetic AD may be different to that seen in non-diabetic AD. The lack of clear functional changes in mitochondrial parameters in diabetic AD suggest different cellular mechanisms contributing to cognitive impairment in diabetes which remain to be fully understood.


Subject(s)
Alzheimer Disease/pathology , Cognitive Dysfunction/pathology , DNA, Mitochondrial/analysis , Diabetes Complications/pathology , Mitochondria/pathology , Aged , Aged, 80 and over , Alzheimer Disease/etiology , Cerebellum/cytology , Cerebellum/pathology , Cognitive Dysfunction/etiology , Cross-Sectional Studies , DNA, Mitochondrial/metabolism , Female , Frontal Lobe/cytology , Frontal Lobe/pathology , Humans , Male , Mitochondria/chemistry , Mitochondria/metabolism , Neurons/cytology , Neurons/pathology , Oxidative Stress , Parietal Lobe/cytology , Parietal Lobe/pathology
13.
FASEB J ; 33(7): 7863-7881, 2019 07.
Article in English | MEDLINE | ID: mdl-30939247

ABSTRACT

Myosteatosis is the pathologic accumulation of lipid that can occur in conjunction with atrophy and fibrosis following skeletal muscle injury. Little is known about the mechanisms by which lipid accumulates in myosteatosis, but many clinical studies have demonstrated that the degree of lipid infiltration negatively correlates with muscle function and regeneration. Our objective was to determine the pathologic changes that result in lipid accumulation in injured muscle fibers. We used a rat model of rotator cuff injury in this study because the rotator cuff muscle group is particularly prone to the development of myosteatosis after injury. Muscles were collected from uninjured controls or 10, 30, or 60 d after injury and analyzed using a combination of muscle fiber contractility assessments, RNA sequencing, and undirected metabolomics, lipidomics, and proteomics, along with bioinformatics techniques to identify potential pathways and cellular processes that are dysregulated after rotator cuff tear. Bioinformatics analyses indicated that mitochondrial function was likely disrupted after injury. Based on these findings and given the role that mitochondria play in lipid metabolism, we then performed targeted biochemical and imaging studies and determined that mitochondrial dysfunction and reduced fatty acid oxidation likely leads to the accumulation of lipid in myosteatosis.-Gumucio, J. P., Qasawa, A. H., Ferrara, P. J., Malik, A. N., Funai, K., McDonagh, B., Mendias, C. L. Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis.


Subject(s)
Adipose Tissue/metabolism , Lipid Metabolism , Mitochondria, Muscle/metabolism , Muscular Disorders, Atrophic/metabolism , Rotator Cuff Injuries/pathology , Adipose Tissue/pathology , Animals , Collagen/analysis , Gene Expression Profiling , Gene Ontology , Lipidomics , Male , Metabolomics , Muscle Contraction , Muscle Denervation , Muscular Disorders, Atrophic/genetics , Muscular Disorders, Atrophic/pathology , Oxidation-Reduction , Principal Component Analysis , Proteomics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Rotator Cuff Injuries/metabolism , Sequence Analysis, RNA
14.
Neurobiol Aging ; 53: 36-47, 2017 05.
Article in English | MEDLINE | ID: mdl-28208064

ABSTRACT

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species.


Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/genetics , Genes, Mitochondrial/genetics , Mitochondria/genetics , Aged , Aged, 80 and over , Alzheimer Disease/diagnosis , Biomarkers/blood , Cognitive Dysfunction/blood , Cognitive Dysfunction/genetics , Female , Gene Expression , Humans , Male , Oxidative Phosphorylation , Reactive Oxygen Species/metabolism , Transcription, Genetic/genetics
15.
Redox Biol ; 10: 100-107, 2016 12.
Article in English | MEDLINE | ID: mdl-27710853

ABSTRACT

Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN.


Subject(s)
Diabetic Nephropathies/metabolism , Hyperglycemia/complications , Kidney Tubules, Proximal/metabolism , Mesangial Cells/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/metabolism , Cell Line , Cell Respiration , Energy Metabolism , Gene Expression Regulation , Glycolysis , Humans , Hyperglycemia/metabolism , Kidney Tubules, Proximal/cytology , Mesangial Cells/cytology
16.
Mitochondrion ; 29: 59-64, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27181048

ABSTRACT

BACKGROUND: Mitochondria contain an extra-nuclear genome in the form of mitochondrial DNA (MtDNA), damage to which can lead to inflammation and bioenergetic deficit. Changes in MtDNA levels are increasingly used as a biomarker of mitochondrial dysfunction. We previously reported that in humans, fragments in the nuclear genome known as nuclear mitochondrial insertion sequences (NumtS) affect accurate quantification of MtDNA. In the current paper our aim was to determine whether mouse NumtS affect the quantification of MtDNA and to establish a method designed to avoid this. METHODS: The existence of NumtS in the mouse genome was confirmed using blast N, unique MtDNA regions were identified using FASTA, and MtDNA primers which do not co-amplify NumtS were designed and tested. MtDNA copy numbers were determined in a range of mouse tissues as the ratio of the mitochondrial and nuclear genome using real time qPCR and absolute quantification. RESULTS: Approximately 95% of mouse MtDNA was duplicated in the nuclear genome as NumtS which were located in 15 out of 21 chromosomes. A unique region was identified and primers flanking this region were used. MtDNA levels differed significantly in mouse tissues being the highest in the heart, with levels in descending order (highest to lowest) in kidney, liver, blood, brain, islets and lung. CONCLUSION: The presence of NumtS in the nuclear genome of mouse could lead to erroneous data when studying MtDNA content or mutation. The unique primers described here will allow accurate quantification of MtDNA content in mouse models without co-amplification of NumtS.


Subject(s)
DNA, Mitochondrial/analysis , Real-Time Polymerase Chain Reaction/methods , Animals , DNA Primers/genetics , DNA, Mitochondrial/genetics , Male , Mice, Inbred C57BL
17.
Diabetes Res Clin Pract ; 110(3): 257-65, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26625720

ABSTRACT

AIMS: We previously showed that circulating mitochondrial DNA (MtDNA) levels are altered in diabetic nephropathy. The aim of the current study was to determine if circulating MtDNA levels are altered in patients with diabetic retinopathy. METHODS: Patients with diabetes (n=220) were studied in a clinical setting using a cross-sectional study design as the following groups: DR-0 (no retinopathy, n=53), DR-m (mild non-proliferative diabetic retinopathy NPDR, n=98) and DR-s (severe proliferative diabetic retinopathy, n=69). MtDNA content in peripheral blood DNA was measured as the mitochondrial to nuclear genome ratio using real time qPCR. Circulating cytokines were measured using the luminex assay and MtDNA damage was assessed using PCR. Differences were considered significant at P<0.05. RESULTS: Circulating MtDNA values were higher in DR-m compared to DR-0 (P=0.02) and decreased in DR-s compared to DR-m (P=0.001). These changes remained significant after adjusting for associated parameters. In parallel there were increased levels of circulating cytokines IL-4 (P=0.005) and TNF-α (P=0.02) in the DR-s group and increased MtDNA damage in DR-m patients compared to DR-0 (P=0.03). CONCLUSIONS: Our data show that circulating MtDNA levels are independently associated with diabetic retinopathy, showing an increase in DR-m and decrease in DR-s with a parallel increase in MtDNA damage and inflammation. Hyperglycemia-induced changes in MtDNA in early diabetes may contribute to inflammation and progression of diabetic retinopathy. Longitudinal studies should be carried out to determine a potential causality of MtDNA in diabetic retinopathy.


Subject(s)
DNA Damage , DNA, Mitochondrial/blood , Diabetic Retinopathy/blood , Inflammation/blood , Adult , Aged , Aged, 80 and over , Cross-Sectional Studies , DNA Copy Number Variations , Diabetes Mellitus, Type 2/blood , Diabetic Retinopathy/complications , Disease Progression , Female , Humans , Hyperglycemia/complications , Inflammation/complications , Interleukin-4/blood , Male , Middle Aged , Risk Factors , Tumor Necrosis Factor-alpha/blood
18.
EBioMedicine ; 2(6): 499-512, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26288815

ABSTRACT

The purpose of this study was to determine if mitochondrial dysfunction plays a role in diabetic nephropathy (DN), a kidney disease which affects > 100 million people worldwide and is a leading cause of renal failure despite therapy. A cross-sectional study comparing DN with diabetes patients without kidney disease (DC) and healthy controls (HCs); and renal mesangial cells (HMCs) grown in normal and high glucose, was carried out. Patients with diabetes (DC) had increased circulating mitochondrial DNA (MtDNA), and HMCs increased their MtDNA within 24 h of hyperglycaemia. The increased MtDNA content in DCs and HMCs was not functional as transcription was unaltered/down-regulated, and MtDNA damage was present. MtDNA was increased in DC compared to HC, conversely, patients with DN had lower MtDNA than DC. Hyperglycaemic HMCs had fragmented mitochondria and TLR9 pathway activation, and in diabetic patients, mitophagy was reduced. Despite MtDNA content and integrity changing within 4 days, hyperglycaemic HMCs had a normal bio-energetic profile until 8 days, after which mitochondrial metabolism was progressively impaired. Peripheral blood mononuclear cells (PBMCs) from DN patients had reduced reserve capacity and maximal respiration, loss of metabolic flexibility and reduced Bioenergetic Health Index (BHI) compared to DC. Our data show that MtDNA changes precede bioenergetic dysfunction and that patients with DN have impaired mitochondrial metabolism compared to DC, leading us to propose that systemic mitochondrial dysfunction initiated by glucose induced MtDNA damage may be involved in the development of DN. Longitudinal studies are needed to define a potential cause-effect relationship between changes in MtDNA and bioenergetics in DN.


Subject(s)
Diabetic Nephropathies/pathology , Hyperglycemia/metabolism , Mesangial Cells/pathology , Mitochondria/pathology , Mitophagy/physiology , Cells, Cultured , Cross-Sectional Studies , DNA, Mitochondrial/blood , DNA, Mitochondrial/genetics , Diabetes Mellitus/pathology , Energy Metabolism/physiology , Enzyme Activation , Glucose/metabolism , Humans , Leukocytes, Mononuclear/physiology , Mitochondria/genetics , Toll-Like Receptor 9/metabolism
20.
Methods Mol Biol ; 1264: 117-31, 2015.
Article in English | MEDLINE | ID: mdl-25631009

ABSTRACT

We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.


Subject(s)
DNA, Mitochondrial/blood , DNA, Mitochondrial/genetics , Real-Time Polymerase Chain Reaction/methods , DNA, Mitochondrial/isolation & purification , Humans , Real-Time Polymerase Chain Reaction/standards , Reference Standards , Reference Values , Reproducibility of Results , Sensitivity and Specificity
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