Anna Czajka

Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction

Mitochondrial dysfunction is central to numerous diseases of oxidative stress. Changes in mitochondrial DNA (MtDNA) content, often measured as mitochondrial genome to nuclear genome ratio (Mt/N) using real time quantitative PCR, have been reported in a broad range of human diseases, such as diabetes and its complications, obesity, cancer, HIV complications, and ageing. We propose the hypothesis that MtDNA content in body fluids and tissues could be a biomarker of mitochondrial dysfunction and review the evidence supporting this theory. Increased reactive oxygen species resulting from an external trigger such as hyperglycaemia or increased fat in conditions of oxidative stress could lead to enhanced mitochondrial biogenesis, and increased Mt/N. Altered MtDNA levels may contribute to enhanced oxidative stress and inflammation and could play a pathogenic role in mitochondrial dysfunction and disease. Changes in Mt/N are detectable in circulating cells such as peripheral blood mononuclear cells and these could be used as surrogate to predict global changes in tissues and organs. We review a large number of studies reporting changes in MtDNA levels in body fluids such as circulating blood cells, cell free serum, saliva, sperm, and cerebrospinal fluid as well as in tumour and normal tissue samples. However, the data are often conflicting as the current methodology used to measure Mt/N can give false results because of one or more of the following reasons (1) use of mitochondrial primers which co-amplify nuclear pseudogenes (2) use of nuclear genes which are variable and/or duplicated in numerous locations (3) a dilution bias caused by the differing genome sizes of the mitochondrial and nuclear genome and (4) template preparation protocols which affect the yields of nuclear and mitochondrial genomes. Development of robust and reproducible methodology is needed to test the hypothesis that MtDNA content in body fluids is biomarker of mitochondrial dysfunction.

Altered Mitochondrial Function, Mitochondrial DNA and Reduced Metabolic Flexibility in Patients With Diabetic Nephropathy

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.

Altered circulating mitochondrial DNA and increased inflammation in patients with diabetic retinopathy

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.

Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences

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.

Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy

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.

Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

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.

Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes.

Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet β-cells and identify the effects of its depletion on β-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 β-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of β-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 β-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to β-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in β-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in β-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in β-cells. These observations demonstrate that ENT3 is predominantly expressed by islet β-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced β-cell apoptosis. Thus, apoptotic loss of islet β-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.

Nop-7-associated 2 (NSA2), a candidate gene for diabetic nephropathy, is involved in the TGFβ1 pathway

We recently showed that Nop-7-associated 2 (NSA2) originally described in yeast as a nuclear protein involved in ribosomal biogenesis, is a hyperglycemia induced gene involved in diabetic nephropathy [Shahni et al., Elevated levels of renal and circulating Nop-7-associated 2 (NSA2) in rat and mouse models of diabetes, in mesangial cells in vitro and in patients with diabetic nephropathy. Diabetologia 2012;55(March(3)):825-34]. However the function of NSA2 in the cell remains unknown. In the current paper we investigate the possible mechanisms for the involvement of NSA2 in diabetic nephropathy by testing the hypothesis that NSA2 expression is linked to the TGFβ1 pathway. Both TGFβ1 and NSA2 mRNAs were significantly up-regulated in cultured renal mesangial cells in response to high glucose, in mouse kidneys during hyperglycemia, and in developing kidneys of mouse embryos during mesenchymal to epithelial transition. Surprisingly, the previously described nuclear NSA2 protein was predominantly located in the cytosol of cultured renal cells. Exogenous TGFβ1 could elevate NSA2 mRNA/protein levels in cultured mesangial cells and could also affect the cellular localization of NSA2, causing the predominantly cytosolic NSA2 protein to rapidly translocate to the nucleus. Increased NSA2 nuclear staining was seen in diabetic mouse kidneys compared to control kidneys. Knock-down of NSA2 expression using RNA interference resulted in significantly decreased TGFβ1 mRNA/protein, almost abolished TGFβ1 activity, and resulted in significantly reduced mRNA levels of the TGFβ1 downstream gene fibronectin. Our data suggest that NSA2 is acting upstream of the TGFβ1 pathway and that NSA2 is needed for TGFβ1 expression and transcriptional activity. In summary, NSA2, which increases in diabetic nephropathy, may be involved in the actions of TGFβ1 and contribute to the development of diabetic nephropathy.

PP011. Placental expression of the major protein components of caveolae, eNOS and iNOS in pre-eclampsia

INTRODUCTION Caveolins and Cavins are the major protein components of caveolae and regulate many cardiovascular functions. Caveolin-1 inhibits eNOS activity. The possible regulation of vascular reactivity/blood pressure by the caveolae are of interest in relation to pre-eclampsia (PE). We hypothesised that expression of Caveolin/Cavin genes would be reduced, paralleling the up-regulated eNOS in PE compared to normotensive controls (NC). OBJECTIVES To analyse the placental mRNA expression of Caveolins1-3 and, Cavins1-4, eNOS and iNOS; and protein of caveolin-1, cavin-1 and eNOS in NC and PE placentae from White European women. METHODS Following ethical approval and informed written consent, placental biopsies were taken midway between the cord and periphery, avoiding infarcts, from 24 NC and 23 PE patients. Gene expression was measured by qRT-PCR. Protein localization was identified by immunohistochemistry and expression semi-quantitatively assessed. RESULTS Results of mRNA/proteins are shown on table below. Protein expression was localised to the cytotrophoblast and syncytiotrophoblast. No differences were found for any other gene/protein. CONCLUSION As well as their known effects on eNOS expression, caveolae mediate internalisation of numerous hormone receptors, thus potentially changing pressor and depressor responsiveness. This is the first time that structural determinants of caveolae have been studied in NC and PE pregnancy. FUNDING Tommys, CAPES.