Karolina Rygiel - Academia.edu (original) (raw)

Papers by Karolina Rygiel

Nucleic acids research, Jan 30, 2016

Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age ... more Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but ∼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evi...

The Journal of Physiology, 2016

Skeletal muscles undergo structural and functional decline with ageing, culminating in sarcopenia... more Skeletal muscles undergo structural and functional decline with ageing, culminating in sarcopenia. The underlying neuromuscular mechanisms have been the subject of intense investigation, revealing mitochondrial abnormalities as potential culprit within both nerve and muscle cells. Implicated mechanisms involve impaired mitochondrial dynamics, reduced organelle biogenesis and quality control via mitophagy, accumulation of mitochondrial DNA (mtDNA) damage and respiratory chain defect, metabolic disturbance, pro-apoptotic signalling, and oxidative stress. This article provides an overview of the cellular mechanisms whereby mitochondria may promote maladaptive changes within motor neurons, the neuromuscular junction (NMJ), and muscle fibres. Lifelong physical activity, which promotes mitochondrial health across tissues, is emerging as an effective countermeasure for sarcopenia. This article is protected by copyright. All rights reserved.

Annals of neurology, Jan 25, 2015

To determine the extent of respiratory chain abnormalities and investigate the contribution of mi... more To determine the extent of respiratory chain abnormalities and investigate the contribution of mitochondrial DNA (mtDNA) to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2 and COXI), and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (Porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number and presence of transcription/replication-associated 7S DNA employing a triplex real-time PCR assay. While mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in t...

Scientific reports, 2015

Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mi... more Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a br...

Journal of neuroscience methods, Jan 30, 2014

Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are als... more Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are also associated with ageing and neurodegenerative disorders, e.g. in substantia nigra (SN) neurons from Parkinson's disease patients. In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Evaluating the full extent of RC deficiency within SN neurons is challenging because their size precludes investigations in serial sections. We developed an assay to measure RC abnormalities in individual SN neurons using quadruple immunofluorescence. Using antibodies against subunits of complex I (CI) and IV, porin and tyrosine hydroxylase together with IgG subtype-specific fluorescent labelled secondary antibodies, we quantified the expression of CI and CIV compared to mitochondrial mass in dopaminergic neurons. CI:porin and CIV:porin ratios were determined relative to a standard control. Quantifica...

PLoS ONE, 2012

Although denervation has long been implicated in aging muscle, the degree to which it is causes t... more Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav 1.5 , in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638634 MNs6mm 21 ) and SEN (469613 MNs6mm 21 ). Nav 1.5 positive fibers (1548670 mm 2 ) were 35% smaller than Nav 1.5 negative fibers (2367678 mm 2 ; P,0.05) in SEN muscle, whereas Nav 1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553633 mm 2 ; P,0.05) where no Nav 1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav 1.5 positive fibers had higher levels of MAFbx and MuRF1 (P,0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.

Neuropathology and Applied Neurobiology, 2014

Sporadic Inclusion Body Myositis (sIBM) is the most common late onset muscle disease causing prog... more Sporadic Inclusion Body Myositis (sIBM) is the most common late onset muscle disease causing progressive weakness. In light of the lack of effective treatment, we investigated potential causes underlying muscle wasting. We hypothesised that accumulation of mitochondrial respiratory deficiency in muscle fibres may lead to fibre atrophy and degeneration, contributing to muscle mass reduction.

Neurobiology of Aging, 2014

Sarcopenia, muscle wasting, and strength decline with age, is an important cause of loss of mobil... more Sarcopenia, muscle wasting, and strength decline with age, is an important cause of loss of mobility in the elderly individuals. The underlying mechanisms are uncertain but likely to involve defects of motor nerve, neuromuscular junction, and muscle. Loss of motor neurons with age and subsequent denervation of skeletal muscle has been recognized as one of the contributing factors. This study investigated aspects of mitochondrial biology in spinal motor neurons from elderly subjects. We found that protein components of complex I of mitochondrial respiratory chain were reduced or absent in a proportion of aged motor neuronsea phenomenon not observed in fetal tissue. Further investigation showed that complex I-deficient cells had reduced mitochondrial DNA content and smaller soma size. We propose that mitochondrial dysfunction in these motor neurons could lead to the cell loss and ultimately denervation of muscle fibers.

Liver Transplantation, 2010

Loss of bile duct epithelium is characteristic of early chronic rejection following liver transpl... more Loss of bile duct epithelium is characteristic of early chronic rejection following liver transplantation. Recent studies have suggested that intrahepatic biliary epithelial cells can transform into myofibroblasts. This study examines the induction and molecular regulation of this transition during allograft rejection. Immortalized human cholangiocytes were stimulated with either transforming growth factor b1 (TGFb1) or a T cell line, and they were examined for morphological, proteomic, and functional features. Posttransplant liver biopsy sections were also examined. Treatment of cholangiocytes with TGFb1 or TGFbpresenting T cells induced a bipolar morphology, reduced expression of E-cadherin and zona occludens 1 (ZO-1), and increased vimentin, fibronectin, matrix metalloproteinase 2 (MMP-2), MMP-9, and S100 calcium binding protein A4 (S100A4); treated cells invaded a model basement membrane. Chemokines induced T cell penetration of 3-dimensional, cultured bile duct-like structures and bile ducts in liver biopsy sections. A spatial association was observed between ductinfiltrating T cells and cholangiocyte expression of mesenchymal markers, including S100A4. Inhibition of S100A4 expression in vitro blocked TGFb1-mediated loss of E-cadherin and ZO-1 but did not reduce induction of fibronectin, MMP-2, or MMP-9. This study demonstrates the potential for T cells to induce an intrahepatic biliary epithelial-to-mesenchymal cell transition during chronic rejection. Furthermore, S100A4 expression by cholangiocytes was identified as a crucial regulator of this transition. Liver Transpl 16:567-576,

Liver International, 2008

Laboratory Investigation, 2008

Journal of Hepatology, 2006

and/or alkaline phosphatases (n 20). The main causes of liver damage (except overweight) were rul... more and/or alkaline phosphatases (n 20). The main causes of liver damage (except overweight) were ruled out. Among them, 26 patients underwent a liver biopsy. Twenty-nine women referred to our hospital for infertility were used as control. All women had karyotype analysis and fluorescence in situ hybridization was performed in case of karyotype abnormality. Results: Overweight (BMI > 25 kg/m 2) was present in 77% of the patients. Abnormal karyotype was observed in 27 patients (88%) and one control (3%, p < 0.0001). The karyotype study revealed X structural abnormalities in 5 patients: translocation in one, X-ring in one and iso-chromosomy of the X long arm in three. Other abnormalities present in 24 patients included complete monosomy in one (46X0) and mosaicism in 23 (45,X0/46,XX or 45X0/46,XX, 47,XXX). We considered the diagnosis of mosaicism when the proportion of X0 cells was equal or superior to 7%. Liver histology was always abnormal and showed isolated or associated changes already described in TS: macrovesicular steatosis (n 20), architectural distortion (n 17), including 12 cases of nodular regenerative hyperplasia, biliary lesions (n 13) including lymphocytic cholangitis (n 9), ductular proliferation (n 8) and ductopenia (n 1). In conclusion, in women with short stature (generally overweighted) and unexplained elevated liver enzymes, chromosome X abnormalities are extremely frequent. The liver lesions observed are similar to those of TS. Anatomical changes of the liver might be a consequence of the deletion of one or more genes located on chromosome X.

American Journal of Respiratory and Critical Care Medicine, 2012

Rationale: Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but t... more Rationale: Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but the cellular basis for this phenomenon, referred to as ventilator-induced diaphragmatic dysfunction (VIDD), is poorly understood. Objectives: To determine whether mitochondrial function and cellular energy status are disrupted in human diaphragms after MV, and the role of mitochondria-derived oxidative stress in the development of VIDD. Methods: Diaphragm and biceps specimens obtained from brain-dead organ donors who underwent MV (15-176 h) and age-matched control subjects were compared regarding mitochondrial enzymatic function, mitochondrial DNA integrity, lipid content, and metabolic gene and protein expression. In addition, diaphragmatic force and oxidative stress after exposure to MV for 6 hours were evaluated in mice under different conditions. Measurements and Main Results: In human MV diaphragms, mitochondrial biogenesis and content were down-regulated, with a more specific defect of respiratory chain cytochrome-c oxidase. Laser capture microdissection of cytochrome-c oxidase-deficient fibers revealed mitochondrial DNA deletions, consistent with damage from oxidative stress. Diaphragmatic lipid accumulation and responses of master cellular metabolic sensors (AMP-activated protein kinase and sirtuins) were consistent with energy substrate excess as a possible stimulus for these changes. In mice, induction of hyperlipidemia worsened diaphragmatic oxidative stress during MV, whereas transgenic overexpression of a mitochondria-localized antioxidant (peroxiredoxin-3) was protective against VIDD. Conclusions: Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.

Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mos... more Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mosaic pattern of deficiency. Biochemical assays are difficult to interpret due to the varying enzyme deficiency levels found in individual cells. Histochemical analysis allows semi-quantitative assessment of complex II and complex IV activities, but there is no validated histochemical assay to assess complex I activity which is frequently affected in mitochondrial pathology. To help improve the diagnosis of mitochondrial disease and to study the mechanisms underlying mitochondrial abnormalities in disease, we have developed a quadruple immunofluorescent technique enabling the quantification of key respiratory chain subunits of complexes I and IV, together with an indicator of mitochondrial mass and a cell membrane marker. This assay gives precise and objective quantification of protein abundance in large numbers of individual muscle fibres. By assessing muscle biopsies from subjects with a range of different mitochondrial genetic defects we have demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle, providing evidence for the diagnostic use of the technique, as well as insight into the underlying molecular pathology. Stringent testing for reproducibility and sensitivity confirms the potential value of the technique for mechanistic studies of disease and in the evaluation of therapeutic approaches. Defects of mitochondrial oxidative phosphorylation (OXPHOS) are found in a wide range of human pathologies, either as a primary cause of disease through genetic defects involving either the mito-chondrial (mtDNA) or nuclear genome 1,2 , or secondary when there are other prominent pathological processes such as inflammation (multiple sclerosis 3 , inclusion body myositis 4) or degenerative features (Parkinson's disease 5). Skeletal muscle is frequently affected by both primary and secondary mitochondrial defects. Mitochondrial myopathies are progressive and clinical features include chronic progressive external oph-thalmoplegia, rhabdomyolysis, muscle fatigue and severe proximal weakness 6. Muscle is also commonly