Institute of Fundamental Technological Research
Polish Academy of Sciences

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Joanna Szczepanowska, PhD, DSc

Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Supervision of doctoral theses
1.  2016-09-30 Walczak Jarosław  
(Instytut Biologii Doświadczalnej PAN)
Zmiany funkcjonowania mitochondriów i organizacji sieci mitochondrialnej w fibroblastach pacjentów ze stwardnieniem bocznym zanikowym (ALS) 

Recent publications
1.  Dębska-Vielhaber G., Miller I., Peeva V., Zuschratter W., Walczak J., Schreiber S., Petri S., Machts J., Vogt S., Szczepanowska J., Gellerich F.N., Hermann A., Vielhaber S., Kunz W.S., Impairment of mitochondrial oxidative phosphorylation in skin fibroblasts of SALS and FALS patients is rescued by in vitro treatment with ROS scavengers, Experimental Neurology, ISSN: 0014-4886, DOI: 10.1016/j.expneurol.2021.113620, Vol.339, pp.113620-1-10, 2021

Abstract:
Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive, neurodegenerative disorder affecting upper and lower motor neurons. Approximately 10% of patients suffer from familial ALS (FALS) with mutations in different ubiquitously expressed genes including SOD1, C9ORF72, TARDBP, and FUS. There is compelling evidence for mitochondrial involvement in the pathogenic mechanisms of FALS and sporadic ALS (SALS), which is believed to be relevant for disease. Owing to the ubiquitous expression of relevant disease-associated genes, mitochondrial dysfunction is also detectable in peripheral patient tissue. We here report results of a detailed investigation of the functional impairment of mitochondrial oxidative phosphorylation (OXPHOS) in cultured skin fibroblasts from 23 SALS and 17 FALS patients, harboring pathogenic mutations in SOD1, C9ORF72, TARDBP and FUS. A considerable functional and structural mitochondrial impairment was detectable in fibroblasts from patients with SALS. Similarly, fibroblasts from patients with FALS, harboring pathogenic mutations in TARDBP, FUS and SOD1, showed mitochondrial defects, while fibroblasts from C9ORF72 associated FALS showed a very mild impairment detectable in mitochondrial ATP production rates only. While we could not detect alterations in the mtDNA copy number in the SALS or FALS fibroblast cultures, the impairment of OXPHOS in SALS fibroblasts and SOD1 or TARDBP FALS could be rescued by in vitro treatments with CoQ10 (5 μM for 3 weeks) or Trolox (300 μM for 5 days). This underlines the role of elevated oxidative stress as a potential cause for the observed functional effects on mitochondria, which might be relevant disease modifying factors.

Keywords:
amyotrophic lateral sclerosis, skin fibroblasts, mitochondrial dysfunction, oxidative stress

Affiliations:
Dębska-Vielhaber G. - Otto-von-Guericke University (DE)
Miller I. - other affiliation
Peeva V. - other affiliation
Zuschratter W. - other affiliation
Walczak J. - IPPT PAN
Schreiber S. - other affiliation
Petri S. - other affiliation
Machts J. - other affiliation
Vogt S. - Otto-von-Guericke University (DE)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Gellerich F.N. - Otto-von-Guericke University (DE)
Hermann A. - other affiliation
Vielhaber S. - Otto-von-Guericke University (DE)
Kunz W.S. - other affiliation
2.  Walczak J., Malińska D., Drabik K., Michalska B., Prill M., Johne S., Luettich K., Szymański J., Peitsch M.C., Hoeng J., Duszyński J., Więckowski M.R., van der Toorn M., Szczepanowska J., Mitochondrial network and biogenesis in response to short and long-term exposure of human BEAS-2B cells to aerosol extracts from the tobacco heating system 2.2, Cellular Physiology and Biochemistry, ISSN: 1015-8987, DOI: 10.33594/000000216, Vol.54, No.2, pp.230-251, 2020

Abstract:
Background/aims: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). Methods: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. Results: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. Conclusion: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

Keywords:
BEAS-2B cells, candidate modified risk tobacco product, cigarette smoke, mitochondrial dynamics, tobacco heating system 2.2.

Affiliations:
Walczak J. - other affiliation
Malińska D. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Drabik K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Michalska B. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Prill M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Johne S. - Philip Morris Products S.A. (CH)
Luettich K. - Philip Morris Products S.A. (CH)
Szymański J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Peitsch M.C. - Philip Morris Products S.A. (CH)
Hoeng J. - Philip Morris Products S.A. (CH)
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Więckowski M.R. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
van der Toorn M. - Philip Morris Products S.A. (CH)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
3.  Walczak J., Dębska-Vielhaber G., Vielhaber S., Szymański J., Charzyńska A., Duszyński J., Szczepanowska J., Distinction of sporadic and familial forms of ALS based on mitochondrial characteristics, The FASEB Journal, ISSN: 0892-6638, DOI: 10.1096/fj.201801843R, Vol.33, No.3, pp.4388-4403, 2019

Abstract:
Bioenergetic failure, oxidative stress, and changes in mitochondrial morphology are common pathologic hallmarks of amyotrophic lateral sclerosis (ALS) in several cellular and animal models. Disturbed mitochondrial physiology has serious consequences for proper functioning of the cell, leading to the chronic mitochondrial stress. Mitochondria, being in the center of cellular metabolism, play a pivotal role in adaptation to stress conditions. We found that mitochondrial dysfunction and adaptation processes differ in primary fibroblasts derived from patients diagnosed with either sporadic or familial forms of ALS. The evaluation of mitochondrial parameters such as the mitochondrial membrane potential, the oxygen consumption rate, the activity and levels of respiratory chain complexes, and the levels of ATP, reactive oxygen species, and Ca2+ show that the bioenergetic properties of mitochondria are different in sporadic ALS, familial ALS, and control groups. Comparative statistical analysis of the data set (with use of principal component analysis and support vector machine) identifies and distinguishes 3 separate groups despite the small number of investigated cell lines and high variability in measured parameters. These findings could be a first step in development of a new tool for predicting sporadic and familial forms of ALS and could contribute to knowledge of its pathophysiology.

Keywords:
amyotrophic lateral sclerosis, neurodegeneration, primary fibroblasts, PCA

Affiliations:
Walczak J. - IPPT PAN
Dębska-Vielhaber G. - Otto-von-Guericke University (DE)
Vielhaber S. - Otto-von-Guericke University (DE)
Szymański J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Charzyńska A. - University of Warsaw (PL)
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
4.  Malińska D., Więckowski M.R., Michalska B., Drabik K., Prill M., Patalas-Krawczyk P., Walczak J., Szymański J., Mathis C., Van der Toorn M., Luettich K., Hoeng J., Peitsch M.C., Duszyński J., Szczepanowska J., Mitochondria as a possible target for nicotine action, Journal of Bioenergetics and Biomembranes, ISSN: 0145-479X, DOI: 10.1007/s10863-019-09800-z, Vol.51, No.4, pp.259-276, 2019

Abstract:
Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

Keywords:
adaptation, mitochondria, nicotine, oxidative stress

Affiliations:
Malińska D. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Więckowski M.R. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Michalska B. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Drabik K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Prill M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Patalas-Krawczyk P. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Walczak J. - IPPT PAN
Szymański J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Mathis C. - Philip Morris Products S.A. (CH)
Van der Toorn M. - Philip Morris Products S.A. (CH)
Luettich K. - Philip Morris Products S.A. (CH)
Hoeng J. - Philip Morris Products S.A. (CH)
Peitsch M.C. - Philip Morris Products S.A. (CH)
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
5.  Malińska D., Szymański J., Patalas-Krawczyk P., Michalska B., Wojtala A., Prill M., Partyka M., Drabik K., Walczak J., Sewer A., Johne S., Luettich K., Peitsch M.C., Hoeng J., Duszyński J., Szczepanowska J., van der Toorn M., Więckowski M.R., Assessment of mitochondrial function following short- and long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product and reference cigarettes, Food and Chemical Toxicology, ISSN: 0278-6915, DOI: 10.1016/j.fct.2018.02.013, Vol.115, pp.1-12, 2018

Abstract:
Mitochondrial dysfunction caused by cigarette smoke is involved in the oxidative stress-induced pathology of airway diseases. Reducing the levels of harmful and potentially harmful constituents by heating rather than combusting tobacco may reduce mitochondrial changes that contribute to oxidative stress and cell damage. We evaluated mitochondrial function and oxidative stress in human bronchial epithelial cells (BEAS 2B) following 1- and 12-week exposures to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), in comparison with TPM from the 3R4F reference cigarette. After 1-week exposure, 3R4F TPM had a strong inhibitory effect on mitochondrial basal and maximal oxygen consumption rates compared to TPM from THS2.2. Alterations in oxidative phosphorylation were accompanied by increased mitochondrial superoxide levels and increased levels of oxidatively damaged proteins in cells exposed to 7.5 μg/mL of 3R4F TPM or 150 μg/mL of THS2.2 TPM, while cytosolic levels of reactive oxygen species were not affected. In contrast, the 12-week exposure indicated adaptation of BEAS-2B cells to long-term stress. Together, the findings indicate that 3R4F TPM had a stronger effect on oxidative phosphorylation, gene expression and proteins involved in oxidative stress than TPM from the candidate modified-risk tobacco product THS2.2.

Keywords:
Mitochondria, Mitochondrial respiratory chain, Oxidative stress, BEAS-2B cells, Cigarette, Tobacco heating system

Affiliations:
Malińska D. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szymański J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Patalas-Krawczyk P. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Michalska B. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Wojtala A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Prill M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Partyka M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Drabik K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Walczak J. - other affiliation
Sewer A. - Philip Morris Products S.A. (CH)
Johne S. - Philip Morris Products S.A. (CH)
Luettich K. - Philip Morris Products S.A. (CH)
Peitsch M.C. - Philip Morris Products S.A. (CH)
Hoeng J. - Philip Morris Products S.A. (CH)
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
van der Toorn M. - Philip Morris Products S.A. (CH)
Więckowski M.R. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
6.  Walczak J., Partyka M., Duszyński J., Szczepanowska J., Implications of mitochondrial network organization in mitochondrial stress signalling in NARP cybrid and Rho0 cells, Scientific Reports, ISSN: 2045-2322, DOI: 10.1038/s41598-017-14964-y, Vol.7, No.14864, pp.1-14, 2017

Abstract:
Mitochondrial dysfunctions lead to the generation of signalling mediators that influence the fate of that organelle. Mitochondrial dynamics and their positioning within the cell are important elements of mitochondria-nucleus communication. The aim of this project was to examine whether mitochondrial shape, distribution and fusion/fission proteins are involved in the mitochondrial stress response in a cellular model subjected to specifically designed chronic mitochondrial stress: WT human osteosarcoma cells as controls, NARP cybrid cells as mild chronic stress and Rho0 as severe chronic stress. We characterized mitochondrial distribution in these cells using confocal microscopy and evaluated the level of proteins directly involved in the mitochondrial dynamics and their regulation. We found that the organization of mitochondria within the cell is correlated with changes in the levels of proteins involved in mitochondrial dynamics and proteins responsible for regulation of this process. Induction of the autophagy/mitophagy process, which is crucial for cellular homeostasis under stress conditions was also shown. It seems that mitochondrial shape and organization within the cell are implicated in retrograde signalling in chronic mitochondrial stress.

Affiliations:
Walczak J. - other affiliation
Partyka M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
7.  Oparka M., Walczak J., Malińska D., van Oppen L.M.P.E., Szczepanowska J., Koopman W.J.H., Więckowski M.R., Quantifying ROS levels using CM-H2DCFDA and HyPer, Methods, ISSN: 1046-2023, DOI: 10.1016/j.ymeth.2016.06.008, Vol.109, pp.3-11, 2016

Abstract:
At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).

Keywords:
Reactive oxygen species, Hydrogen peroxide, CM-H2DCFDA, HyPer, SypHer

Affiliations:
Oparka M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Walczak J. - other affiliation
Malińska D. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
van Oppen L.M.P.E. - RIMLS, RCMM, Radboudumc (NL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Koopman W.J.H. - RIMLS, RCMM, Radboudumc (NL)
Więckowski M.R. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
8.  Wojewoda M., Walczak J., Duszyński J., Szczepanowska J., Selenite activates the ATM kinase-dependent DNA repair pathway in human osteosarcoma cells with mitochondrial dysfunction, Biochemical Pharmacology, ISSN: 0006-2952, DOI: 10.1016/j.bcp.2015.03.016, Vol.95, No.3, pp.170-176, 2015

Abstract:
Mitochondrial dysfunction and reactive oxygen species (ROS) induced oxidative damage are implicated in the pathogenesis of several human diseases. Based on our previous findings that ROS level was higher in human osteosarcoma cybrids—Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) and was reduced by selenite treatment, this study was designed to elucidate the effects of selenite administration on oxidative and nitrosative damage to lipids, proteins and DNA.

Oxidative and nitrosative damage to lipids and proteins was not increased in NARP cybrids or mitochondrial DNA-lacking Rho0 cells (displaying mitochondrial dysfunction) when compared with control WT cells. However, we found the enhanced formation of DNA double-strand breaks based on the level of histone γH2AX (phosphorylated at Ser 139), which is known to be phosphorylated by ATM (Ataxia Telangiectasia Mutated) kinase in response to DNA damage. Selenite increased the activity of ATM kinase in NARP cybrids and Rho0 cells without concomitant increase in levels of histone γH2AX.

Activation of the ATM kinase-dependent DNA repair pathway triggered by selenite could not be associated with enhanced DNA damage but might rather result from selenite-induced activation of ATM-dependent DNA repair mechanisms which could account for protective effects of this agent.

Keywords:
Mitochondrial dysfunction, Selenite, DNA repair, ATM kinase, Oxidative damage

Affiliations:
Wojewoda M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Walczak J. - other affiliation
Duszyński J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
9.  Walczak J., Szczepanowska J., Zaburzenia dynamiki i dystrybucji mitochondriów w komórkach w stwardnieniu zanikowym bocznym (ALS), Postępy Biochemii, ISSN: 0032-5422, Vol.61, No.2, pp.183-190, 2015

Abstract:
Stwardnienie zanikowe boczne (ALS) jest chorobą o złożonej etiologii, prowadzącą do degradacji neuronów ruchowych. Jednym z pierwszych objawów w rozwoju wielu chorób neurodegeneracyjnych, m. in. w ALS, są zaburzenia funkcjonowania mitochondriów. Już kilka dekad temu obserwowano zmiany morfologii mitochondriów w tkankach pacjentów cierpiących na to schorzenie. Mitochondria są organellami dynamicznymi, ulegają ciągłym procesom fuzji i fragmentacji oraz przemieszczania się w komórce. Prawidłowy przebieg procesów związanych z dynamiką i dystrybucją mitochondriów jest kluczowy dla funkcjonowania komórek, a w szczególności komórek nerwowych o silnie wydłużonych aksonach. Praca ta stanowi podsumowanie istniejącej wiedzy na temat roli dynamiki i dystrybucji mitochondriów w patofizjologii ALS, formy rodzinnej i sporadycznej.

Keywords:
ALS, dynamika mitochondriów, transport mitochondriów, neurodegeneracja

Affiliations:
Walczak J. - other affiliation
Szczepanowska J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

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