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Interplay of ionizing radiation, oxygen, ROS and age-associated diseases

Džinić, Tamara :
Interplay of ionizing radiation, oxygen, ROS and age-associated diseases.
[Online-Edition: http://tuprints.ulb.tu-darmstadt.de/6275]
Technische Universität , Darmstadt
[Dissertation], (2017)

Offizielle URL: http://tuprints.ulb.tu-darmstadt.de/6275

Kurzbeschreibung (Abstract)

The aim of this doctoral thesis was to investigate and to understand the implication of oxidative stress, created by the interplay of ionizing (X-ray) radiation, oxygen and neurotoxic amyloid beta (Aβ) peptide, in age-associated diseases, with the focus on Alzheimer´s disease (AD), the most common dementia. Although AD has been known for more than hundred years, its mechanisms are still intriguing and there is no cure at the moment. High doses of ionizing radiation (IR) lead to learning and memory impairment which is characteristic for AD as well. The cumulative doses of IR used in medical imaging procedures such as computed tomography (CT) and dental X-rays present a potential danger, particularly to children whose brains are not completely developed. Although single doses of radiation used for diagnostic purposes or therapeutic treatment are relatively low, small changes on the molecular and cellular level may accumulate upon repeated exposure and result in delayed long-term defects. Thus, the most important objective of this thesis was to reveal if there is an interplay of radiation and age-associated diseases through the effects of oxygen, reactive oxygen species (ROS) and Aβ peptide implicated in AD. Aβ peptide monomers and small oligomers (one of the players in AD) are proposed to be involved in damage and death of neurons. Since IR causes oxidative stress and inflammation, occurring in AD as well, there is a concern that radiation exposure may be linked with neurodegeneration. 2 Gy X-rays presents a commonly used fractionated dose in radiotherapy of a variety of tumors and was used in this thesis as a single radiation dose alone or combined with the previous treatment with externally applied Aβ1-42 peptide (the most toxic form) at two environmental oxygen concentrations. The data provided in the thesis emphasize the importance of using the appropriate model system and conditions in the cell culture such as oxygen concentration that has to be considered in studies of cellular responses to oxidative stress, IR and neurotoxic peptides. Changes in cellular responses of non-differentiated human neuroblastoma (SH-SY5Y) cells and/or cells pre-treated with retinoic acid (RA) for induction of differentiation, in order to obtain cells that resemble neurons, were investigated in parallel under standard condition of oxygen (~21%) used in cell culture incubators, but never found in tissues of human body, and 5% O2 which resembles physiological oxygen concentration in human brain. For this purpose, home-made low O2 incubators were setup using plastic containers able to retain a gas mixture containing 5% or 1% O2, 5% CO2 and 90% or 94% N2. Initially, two different low oxygen concentrations (1% and 5%) were tested in SH-SY5Y cells for cell proliferation and ATP concentration as a measure of the bioenergetic status. 5% O2 was demonstrated to be more suitable for cultivation of SH-SY5Y cell since the proliferation activity and ATP concentration were higher than in cells at 1% O2. The starting hypothesis was that cells exposed to non-physiological oxygen concentration and/or in the presence of Aβ peptide react differently, more sensitive or less sensitive, due to the oxygen effect (i.e. increased sensitivity due to the increased formation of harmful ROS and modulation of signaling pathways or decreased sensitivity due to the previous adaptation to these processes). A very important result was that the oxygen concentration in the cell culture and differentiation status of SH-SY5Y cells are modulators of cellular responses to X-ray radiation and Aβ peptide as shown employing cell and molecular biology as well as biochemistry methods and techniques. Retinoic acid used for induction of differentiation leads to morphological changes of SH-SY5Y cells (i.e. flattening of the cell body and formation of long outgrowths that resemble axons of neurons) and increase in the sensitivity to radiation and/or to Aβ peptide depending on oxygen concentration. Moreover, oxygen plays a role in differentiation and proliferation of SH-SY5Y cells since the amount of neurofilament-M, a marker of differentiation, was dependent of oxygen concentration and was higher in cells cultivated at 5% O2 which also showed increased cell number in the proliferation assay as compared to 21% O2. Intracellular oxygen concentration [O2] differs from the oxygen concentration applied to the cell culture. Using an oxygen sensitive probe, intracellular [O2] for SH-SY5Y cells cultivated at 21% O2 was about 10% and for cells cultivated at 5% O2 was 0.9% determined for the first time. However, 5% O2 was not hypoxic for SH-SY5Y cells since no expression of hypoxia inducible factor (HIF-1α) was detected. Changes in the mitochondrial proteome isolated from SH-SY5Y cells were investigated using a combination of blue-native/2D SDS-PAGE for protein separation, subsequent staining with the fluorescent dye SYPRO Ruby and quantitation of protein spot intensities from gel images. The protein amount of selected subunits of Oxidative Phosphorylation (OxPhos) complexes and other non-OxPhos mitochondrial proteins (identified by MALDI-TOF/TOF mass spectrometry and Western blot) did not change upon irradiation with the exception of the heat shock protein 70, which amount was increased depending on oxygen concentration and differentiation status. Monomers and small oligomers of Aβ peptide interacted with SH-SY5Y cells as detected by flow cytometry. Compartments with low pH value (i.e. lysosomes and possibly late endosomes) were the main target and the peptide interacted only to a minor extent with mitochondria and endoplasmatic reticulum, respectively, as visualized by confocal scanning microscopy. Although site and kinetics of interaction of Aβ peptide with SH-SY5Y cells cultivated at both 21% and 5% O2 were similar, decreased lysosomal integrity after Aβ peptide treatment of SH-SY5Y cells was observed solely at 21% O2. Aβ peptide alone induced only a slight increase in ROS (up to 1.2 fold), a slight increase in mitochondrial membrane potential, and no changes in ATP concentration and in glutathione (GSH) level (used as an indicator of cell´s antioxidant capacity) regardless of oxygen concentration in the cell culture. Radiation alone led to a significant increase in ROS (~1.5 fold) and a slight increase in ATP concentration (~1.2 fold) but solely in cells at 5% O2. A slight increase in mitochondrial membrane potential was observed at both 21% and 5% O2. Radiation caused a slight increase in GSH level at 21% O2 and decrease at 5% O2 (up to 1.2 fold). Increase in protein carbonylation (~2.5 fold) induced by oxidative stress measured in the Oxyblot assay was specific for irradiated cells at 21% O2. A very important target of oxidative stress is DNA, particularly mtDNA that is less studied than nuclear DNA. MtDNA was assayed using primers specific for mtDNA in PCR methods for changes in its amount and for the presence of 4977 bp deletion. Aß peptide or radiation alone caused a change in mtDNA amount depending on O2 concentration: up to 1.3 fold decrease at 21% O2 and up to 1.5 fold increase at 5% O2. The occurrence of the mtDNA deletion was specific for irradiated cells at both 21% and 5% O2 but was more pronounced at 21% O2. For the first time, the combined effect of Aβ peptide and IR on cellular parameters and survival was studied. Radiation combined with Aβ peptide resulted in a statistically significant increase in ROS level at both 21% (~1.2 fold) and 5% O2 (~1.4 fold); increase in ATP concentration (1.5 fold) at 5% O2 solely; increase in GSH level at 21% O2 and decrease at 5% O2 (up to 1.2 fold) and no change in the mitochondrial membrane potential. The results of the cell death assay (measurement of apoptotic and necrotic cells by flow cytometry) revealed that Aβ1-42 peptide or 2 Gy X-rays alone resulted in a significant increase in cell death of SH-SY5Y cells at 5% O2 and only in a minor increase at 21% O2. Noteworthy, Aβ peptide restored the cell death of irradiated cells to the control level or below, particularly at 5% O2. The combination of Aβ peptide and irradiation did not lead to a significant increase in protein carbonylation, mtDNA deletion and change in mtDNA amount. Therefore, the initial level of oxidative stress determines the point at which cellular defense mechanisms occur and it is possible that even neuroprotective mechanisms are triggered in such cases. Furthermore, this study demonstrated that the incubation time together with concentration and state of the peptide (disaggregated versus aggregated to fibrils) is a crucial factor in Aβ peptide toxicity. High concentration (100 µM) of aggregated peptide (induced by storage at 37 °C overnight) caused an early response (after 1 day) of SH-SY5Y cells which underwent cell death. However, the effect of disaggregated Aß peptide (100 µM) after 3 days of incubation was more pronounced than of aggregated peptide. Aβ peptide treatment decreased the percentage of apoptotic and necrotic cells (2.5 fold) in irradiated cells after 1 day. However, after 3 days, a ~2.8 fold increase in cell death as compared to non-irradiated control cells was observed. The observed traffic of Aβ peptide towards cellular organelles and the corresponding changes in cell physiology that were dependent on the level of oxidative stress are of relevance for AD pathology. The results obtained require the use of more physiological 5% O2 rather than of the non-physiological 21% O2 for cultivation of SH-SY5Y cells and studies of their response to stress, which is crucial for outcome and reliability of experiments. Cells cultivated at 5% O2 have displayed higher total cellular ATP concentration (~1.3 fold), lower ROS level (1.5 fold), lower protein carbonlyation (2.5 fold), lower mtDNA deletion (2 fold) as compared to cells at 21% O2.

Typ des Eintrags: Dissertation
Erschienen: 2017
Autor(en): Džinić, Tamara
Titel: Interplay of ionizing radiation, oxygen, ROS and age-associated diseases
Sprache: Englisch
Kurzbeschreibung (Abstract):

The aim of this doctoral thesis was to investigate and to understand the implication of oxidative stress, created by the interplay of ionizing (X-ray) radiation, oxygen and neurotoxic amyloid beta (Aβ) peptide, in age-associated diseases, with the focus on Alzheimer´s disease (AD), the most common dementia. Although AD has been known for more than hundred years, its mechanisms are still intriguing and there is no cure at the moment. High doses of ionizing radiation (IR) lead to learning and memory impairment which is characteristic for AD as well. The cumulative doses of IR used in medical imaging procedures such as computed tomography (CT) and dental X-rays present a potential danger, particularly to children whose brains are not completely developed. Although single doses of radiation used for diagnostic purposes or therapeutic treatment are relatively low, small changes on the molecular and cellular level may accumulate upon repeated exposure and result in delayed long-term defects. Thus, the most important objective of this thesis was to reveal if there is an interplay of radiation and age-associated diseases through the effects of oxygen, reactive oxygen species (ROS) and Aβ peptide implicated in AD. Aβ peptide monomers and small oligomers (one of the players in AD) are proposed to be involved in damage and death of neurons. Since IR causes oxidative stress and inflammation, occurring in AD as well, there is a concern that radiation exposure may be linked with neurodegeneration. 2 Gy X-rays presents a commonly used fractionated dose in radiotherapy of a variety of tumors and was used in this thesis as a single radiation dose alone or combined with the previous treatment with externally applied Aβ1-42 peptide (the most toxic form) at two environmental oxygen concentrations. The data provided in the thesis emphasize the importance of using the appropriate model system and conditions in the cell culture such as oxygen concentration that has to be considered in studies of cellular responses to oxidative stress, IR and neurotoxic peptides. Changes in cellular responses of non-differentiated human neuroblastoma (SH-SY5Y) cells and/or cells pre-treated with retinoic acid (RA) for induction of differentiation, in order to obtain cells that resemble neurons, were investigated in parallel under standard condition of oxygen (~21%) used in cell culture incubators, but never found in tissues of human body, and 5% O2 which resembles physiological oxygen concentration in human brain. For this purpose, home-made low O2 incubators were setup using plastic containers able to retain a gas mixture containing 5% or 1% O2, 5% CO2 and 90% or 94% N2. Initially, two different low oxygen concentrations (1% and 5%) were tested in SH-SY5Y cells for cell proliferation and ATP concentration as a measure of the bioenergetic status. 5% O2 was demonstrated to be more suitable for cultivation of SH-SY5Y cell since the proliferation activity and ATP concentration were higher than in cells at 1% O2. The starting hypothesis was that cells exposed to non-physiological oxygen concentration and/or in the presence of Aβ peptide react differently, more sensitive or less sensitive, due to the oxygen effect (i.e. increased sensitivity due to the increased formation of harmful ROS and modulation of signaling pathways or decreased sensitivity due to the previous adaptation to these processes). A very important result was that the oxygen concentration in the cell culture and differentiation status of SH-SY5Y cells are modulators of cellular responses to X-ray radiation and Aβ peptide as shown employing cell and molecular biology as well as biochemistry methods and techniques. Retinoic acid used for induction of differentiation leads to morphological changes of SH-SY5Y cells (i.e. flattening of the cell body and formation of long outgrowths that resemble axons of neurons) and increase in the sensitivity to radiation and/or to Aβ peptide depending on oxygen concentration. Moreover, oxygen plays a role in differentiation and proliferation of SH-SY5Y cells since the amount of neurofilament-M, a marker of differentiation, was dependent of oxygen concentration and was higher in cells cultivated at 5% O2 which also showed increased cell number in the proliferation assay as compared to 21% O2. Intracellular oxygen concentration [O2] differs from the oxygen concentration applied to the cell culture. Using an oxygen sensitive probe, intracellular [O2] for SH-SY5Y cells cultivated at 21% O2 was about 10% and for cells cultivated at 5% O2 was 0.9% determined for the first time. However, 5% O2 was not hypoxic for SH-SY5Y cells since no expression of hypoxia inducible factor (HIF-1α) was detected. Changes in the mitochondrial proteome isolated from SH-SY5Y cells were investigated using a combination of blue-native/2D SDS-PAGE for protein separation, subsequent staining with the fluorescent dye SYPRO Ruby and quantitation of protein spot intensities from gel images. The protein amount of selected subunits of Oxidative Phosphorylation (OxPhos) complexes and other non-OxPhos mitochondrial proteins (identified by MALDI-TOF/TOF mass spectrometry and Western blot) did not change upon irradiation with the exception of the heat shock protein 70, which amount was increased depending on oxygen concentration and differentiation status. Monomers and small oligomers of Aβ peptide interacted with SH-SY5Y cells as detected by flow cytometry. Compartments with low pH value (i.e. lysosomes and possibly late endosomes) were the main target and the peptide interacted only to a minor extent with mitochondria and endoplasmatic reticulum, respectively, as visualized by confocal scanning microscopy. Although site and kinetics of interaction of Aβ peptide with SH-SY5Y cells cultivated at both 21% and 5% O2 were similar, decreased lysosomal integrity after Aβ peptide treatment of SH-SY5Y cells was observed solely at 21% O2. Aβ peptide alone induced only a slight increase in ROS (up to 1.2 fold), a slight increase in mitochondrial membrane potential, and no changes in ATP concentration and in glutathione (GSH) level (used as an indicator of cell´s antioxidant capacity) regardless of oxygen concentration in the cell culture. Radiation alone led to a significant increase in ROS (~1.5 fold) and a slight increase in ATP concentration (~1.2 fold) but solely in cells at 5% O2. A slight increase in mitochondrial membrane potential was observed at both 21% and 5% O2. Radiation caused a slight increase in GSH level at 21% O2 and decrease at 5% O2 (up to 1.2 fold). Increase in protein carbonylation (~2.5 fold) induced by oxidative stress measured in the Oxyblot assay was specific for irradiated cells at 21% O2. A very important target of oxidative stress is DNA, particularly mtDNA that is less studied than nuclear DNA. MtDNA was assayed using primers specific for mtDNA in PCR methods for changes in its amount and for the presence of 4977 bp deletion. Aß peptide or radiation alone caused a change in mtDNA amount depending on O2 concentration: up to 1.3 fold decrease at 21% O2 and up to 1.5 fold increase at 5% O2. The occurrence of the mtDNA deletion was specific for irradiated cells at both 21% and 5% O2 but was more pronounced at 21% O2. For the first time, the combined effect of Aβ peptide and IR on cellular parameters and survival was studied. Radiation combined with Aβ peptide resulted in a statistically significant increase in ROS level at both 21% (~1.2 fold) and 5% O2 (~1.4 fold); increase in ATP concentration (1.5 fold) at 5% O2 solely; increase in GSH level at 21% O2 and decrease at 5% O2 (up to 1.2 fold) and no change in the mitochondrial membrane potential. The results of the cell death assay (measurement of apoptotic and necrotic cells by flow cytometry) revealed that Aβ1-42 peptide or 2 Gy X-rays alone resulted in a significant increase in cell death of SH-SY5Y cells at 5% O2 and only in a minor increase at 21% O2. Noteworthy, Aβ peptide restored the cell death of irradiated cells to the control level or below, particularly at 5% O2. The combination of Aβ peptide and irradiation did not lead to a significant increase in protein carbonylation, mtDNA deletion and change in mtDNA amount. Therefore, the initial level of oxidative stress determines the point at which cellular defense mechanisms occur and it is possible that even neuroprotective mechanisms are triggered in such cases. Furthermore, this study demonstrated that the incubation time together with concentration and state of the peptide (disaggregated versus aggregated to fibrils) is a crucial factor in Aβ peptide toxicity. High concentration (100 µM) of aggregated peptide (induced by storage at 37 °C overnight) caused an early response (after 1 day) of SH-SY5Y cells which underwent cell death. However, the effect of disaggregated Aß peptide (100 µM) after 3 days of incubation was more pronounced than of aggregated peptide. Aβ peptide treatment decreased the percentage of apoptotic and necrotic cells (2.5 fold) in irradiated cells after 1 day. However, after 3 days, a ~2.8 fold increase in cell death as compared to non-irradiated control cells was observed. The observed traffic of Aβ peptide towards cellular organelles and the corresponding changes in cell physiology that were dependent on the level of oxidative stress are of relevance for AD pathology. The results obtained require the use of more physiological 5% O2 rather than of the non-physiological 21% O2 for cultivation of SH-SY5Y cells and studies of their response to stress, which is crucial for outcome and reliability of experiments. Cells cultivated at 5% O2 have displayed higher total cellular ATP concentration (~1.3 fold), lower ROS level (1.5 fold), lower protein carbonlyation (2.5 fold), lower mtDNA deletion (2 fold) as compared to cells at 21% O2.

Ort: Darmstadt
Fachbereich(e)/-gebiet(e): 07 Fachbereich Chemie > Fachgebiet Biochemie
Hinterlegungsdatum: 04 Jun 2017 19:55
Offizielle URL: http://tuprints.ulb.tu-darmstadt.de/6275
URN: urn:nbn:de:tuda-tuprints-62751
Gutachter / Prüfer: Dencher, Prof. Dr. Norbert A. ; Meckel, PD Dr. Tobias
Datum der Begutachtung bzw. der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 15 Mai 2017
Alternatives oder übersetztes Abstract:
AbstractSprache
Ziel dieser Doktorarbeit war es den Einfluß von oxidativem Stress zu verstehen, der durch das Zusammenspiel von ionisierender Strahlung, Sauerstoff und neurotoxischem Amyloid beta (Aβ) Peptid der Alzheimer Demenz (AD) entstanden ist. Obwohl AD, die häufigste Form von Demenz, seit mehr als hundert Jahren bekannt ist, sind seine Mechanismen noch nicht verstanden und es gibt zur Zeit keine Heilung. Hohe Dosen ionisierender Strahlung führen zu Lern- und Gedächtnisstörungen, die auch für AD charakteristisch sind. Die kumulativen Dosen von ionisierender Strahlung, die in medizinischen Bildgebungsverfahren wie Computertomographie und zahnärztlichen Untersuchungen verwendet werden, stellen eine potentielle Gefahr dar, insbesondere für Kinder, deren Gehirn nicht vollständig entwickelt ist. Obwohl einzelne Dosen von Strahlung, die für diagnostische Zwecke oder Behandlung verwendet werden, relativ niedrig sind, können kleine Schäden auf molekularer und zellulärer Ebene bei wiederholter Exposition akkumulieren und zu verzögerten Langzeitdefekten führen. So war das wichtigste Ziel dieser Arbeit zu zeigen, ob es Wechselwirkungen von Strahlung und altersbedingten Erkrankungen durch die Effekte von Sauerstoff, reaktiver Sauerstoffspezies (ROS) und Aβ Peptid gibt, die an AD beteiligt sind. Aβ Peptid Monomere und kleine Oligomere (Akteure in AD) sollen an Schäden und Tod von Neuronen beteiligt sein. Da ionisierende Strahlung oxidativen Stress und Entzündungen verursacht, die auch in AD auftreten, besteht die Möglichkeit, dass Strahlenbelastung mit Neurodegeneration verknüpft ist. 2 Gy Röntgenstrahlen ist eine häufig verwendete Dosis in der Strahlentherapie einer Vielzahl von Tumoren und wurde in dieser Arbeit als Strahlendosis alleine oder in Kombination mit der vorherigen Behandlung mit extern applizierten Aβ1-42 Peptid (die schädlichste Form) bei zwei unterschiedlichen Sauerstoffkonzentrationen eingesetzt. Die in der Arbeit erhaltenen Ergebnisse betonen die Bedeutung von geeigneten Zellkulturbedingungen, insbesondere der Sauerstoffkonzentration, die in Studien der zellulären Reaktionen auf oxidativen Stress, ionisierende Strahlung und neurotoxischem Peptid eingesetzt werden müssen. Änderungen der zellulären Reaktionen von nicht-differenzierten menschlichen Neuroblastom (SH-SY5Y) Zellen und von mit Retinsäure (RA) zur Induktion der Differenzierung vorbehandelten Zellen, die Neuronen ähneln, wurden unter drei Sauerstoffkonzentrationen parallel untersucht. Bei ~21% Sauerstoff, was gewöhnlich in Zellkultur-Inkubatoren verwendet wird, aber nie in Geweben des menschlichen Körpers vorkommt und bei 5% bzw. 1% O2, was der physiologischen Sauerstoffkonzentration im menschlichen Gehirn ähnelt. Zu diesem Zweck wurden O2-Inkubatoren aus Kunststoffbehältern angefertigt, in denen die Zellen in einem Gasgemisch mit 5% oder 1% O2, 5% CO2 und 90% oder 94% N2 kultiviert werden konnten. Anfänglich wurden die Auswirkungen von zwei verschiedenen niedrigen Sauerstoffkonzentrationen (1% und 5%) in SH-SY5Y Zellen auf die Zellproliferation und ATP-Konzentration als Maß für den bioenergetischen Status getestet. 5% O2 war für die Kultivierung der SH-SY5Y Zellen besser geeignet, da die Proliferationsaktivität und die ATP-Konzentration höher waren als bei den Zellen mit 1% O2. Die Ausgangshypothese war, dass Zellen, die einer nicht-physiologischen hohen Sauerstoffkonzentration ausgesetzt sind, gegenüber Strahlung und/oder dem Vorhandensein von Aβ Peptid aufgrund des Sauerstoffeffekts anders reagieren, empfindlicher oder unempfindlicher sind (d.h. erhöhte Empfindlichkeit aufgrund der erhöhten Bildung von schädlichem ROS und Modulation von Signalwegen oder aber unempfindlicher wegen vorheriger Adaptation dieser Prozesse). Ein sehr wichtiges Ergebnis war, dass die Sauerstoffkonzentration in der Zellkultur und der Differenzierungsstatus von SH-SY5Y Zellen wichtige Modulatoren von zellulären Reaktionen auf Röntgenstrahlung und Aβ Peptid sind. Dies ergab sich aus den zell- und molekularbiologischen sowie aus den biochemischen Untersuchungen. Die zur Induktion der Differenzierung verwendete Retinsäure führt zu morphologischen Veränderungen von SH-SY5Y Zellen (d.h. Abflachung des Zellkörpers und Bildung von langen Auswüchsen, die Axonen von Neuronen ähneln) und zu einer Erhöhung der Empfindlichkeit gegenüber Strahlung und Aβ Peptid in Abhängigkeit von der Sauerstoffkonzentration. Darüber hinaus spielt Sauerstoff eine Rolle bei der Differenzierung und Proliferation von SH-SY5Y Zellen, da die Menge an Neurofilament-M, ein Marker der Differenzierung, von der Sauerstoffkonzentration abhängig ist und bei 5% O2 kultivierten Zellen höher war. Der Proliferationstest zeigte ebenfalls eine erhöhte Zellzahl im Vergleich zu den kultivierten Zellen bei 21% O2. Unter Verwendung einer sauerstoffempfindlichen optischen Sonde konnte nachgewiesen werden, dass die intrazelluläre Sauerstoffkonzentration [O2] sich von der externen Sauerstoffkonzentration unterscheidet. Die intrazelluläre [O2] von SH-SY5Y Zellen, die bei 21% O2 kultiviert wurden, war 10%, die bei 5% O2 kultivierten dagegen 0,9% O2. 5% externe Sauerstoffkonzentration ist für SH-SY5Y Zellen nicht hypoxisch, da keine Expression von Hypoxie-induzierbarem Faktor (HIF-1α) nachgewiesen wurde. Veränderungen des aus SH-SY5Y Zellen isolierten mitochondrialen Proteoms wurden unter Verwendung einer Kombination von blau-nativer/2D-SDS-PAGE zur Proteintrennung, anschließende Färbung mit dem Fluoreszenzfarbstoff SYPRO Ruby und Quantifizierung von Proteinspotsintensitäten aus Gelbildern untersucht. Die Proteinmenge ausgewählter Untereinheiten von Enzymkomplexen der oxidativen Phosphorylierung (OxPhos) und anderer nicht-OxPhos-mitochondrialer Proteine (identifiziert durch MALDI-TOF/TOF-Massenspektrometrie und Western Blot) änderte sich bei der Bestrahlung nicht. Nur die Menge des Hitzeschockproteins 70 erhöhte sich in Abhängigkeit von der Sauerstoffkonzentration und dem Differenzierungsstatus. Monomere und kleine Oligomere des Aβ Peptids interagierten mit SH-SY5Y Zellen, wie durch Durchflusszytometrie nachgewiesen wurde. Kompartimente mit niedrigem pH-Wert (d.h. Lysosomen und möglicherweise späte Endosomen) waren der Hauptzielort (Target) des Peptids während es nur in geringem Maße mit Mitochondrien und dem endoplasmatischen Retikulum in Wechselwirkung tritt, wie durch konfokale Rastermikroskopie sichtbar gemacht wurde. Obwohl der Ort und die Kinetik der Wechselwirkung von Aβ Peptid mit SH-SY5Y Zellen, die bei 21% oder bei 5% O2 kultiviert wurden, ähnlich war, wurde eine verminderte lysosomale Integrität nach der Aβ Peptidbehandlung von SH-SY5Y Zellen allein bei 21% O2 beobachtet. Aβ Peptid allein induzierte nur eine leichte Erhöhung von ROS (bis zu 1,2-fach), eine leichte Zunahme des mitochondrialen Membranpotentials und keine Änderungen in der ATP Konzentration und in der Menge von Glutathion (GSH) (ein Indikator für die Antioxidationskapazität der Zelle), unabhängig von der Sauerstoffkonzentration in der Zellkultur. Strahlung allein führte zu einem signifikanten Anstieg der ROS (~ 1,5-fach) und einer leichten Erhöhung der ATP-Konzentration (~1,2-fach), aber nur in Zellen bei 5% O2. Eine leichte Zunahme des mitochondrialen Membranpotentials wurde bei 21% und 5% O2 beobachtet. Strahlung bewirkte eine leichten Anstieg der GSH-Menge bei 21% O2 und eine Abnahme bei 5% O2 (bis zu 1,2-fach). Die Zunahme der Proteincarbonylierung (~ 2,5-fach) durch oxidativen Stress, gemessen im Oxyblot-Assay, war für bestrahlte Zellen bei 21% O2 spezifisch. Ein sehr sensitiver Wirkungsort des oxidativen Stresses ist DNA, insbesondere mtDNA, die aber bis jetzt viel weniger untersucht wird als nukleare DNA. MtDNA wurde unter Verwendung von für mtDNA spezifischen Primern in PCR-Verfahren auf Änderungen in seiner Menge und auf das Vorhandensein von 4977 bp-Deletion untersucht. Aß Peptid oder Strahlung allein führen zu einer Veränderung der mtDNA Menge in Abhängigkeit von der O2 Konzentration: bis zu 1,3-fache Abnahme bei 21% O2 und bis zu 1,5-fachem Anstieg bei 5% O2. Das Auftreten der mtDNA-Deletion war für bestrahlte Zellen sowohl bei 21% als auch bei 5% O2 spezifisch, war aber bei 21% O2 stärker ausgeprägt. Zum ersten Mal wurde die kombinierte Wirkung von Aβ Peptid und ionisierender Strahlung auf zelluläre Parameter und Zellüberleben untersucht. Die mit Aβ Peptid kombinierte Strahlung führte zu einem statistisch signifikanten Anstieg der ROS-Menge bei 21% (~1,2-fach) und 5% O2 (~1,4-fach), Erhöhung der ATP-Konzentration (1,5-fach) nur bei 5% O2, Erhöhung der GSH-Menge bei 21% O2 und Abnahme bei 5% O2 (bis zu 1,2 fach) und zu keiner Veränderung des mitochondrialen Membranpotentials. Die Ergebnisse des Zelltod-Assays (Messung von apoptotischen und nekrotischen Zellen durch Durchflusszytometrie) zeigten, dass Aβ1-42 Peptid oder 2 Gy-Röntgenstrahlen allein zu einer signifikanten Zunahme des Zelltods von SH-SY5Y Zellen bei 5% O2 führte und nur in einer geringfügigen Zunahme bei 21% O2. Bemerkenswerterweise verringerte Aβ Peptid den Zelltod von bestrahlten Zellen auf das Kontrollniveau oder darunter, insbesondere bei 5% O2. Im Gegensatz zu den individuellen Stressoren führte die Kombination von Aβ Peptid und Bestrahlung nicht zu einer signifikanten Zunahme der Proteincarbonylierung, der mtDNA Deletion und der Änderung der mtDNA Menge. Daher bestimmt der anfängliche Oxidationsstress den Punkt, an dem zelluläre Abwehrmechanismen auftreten, und es ist möglich, dass in diesem Fall auch neuroprotektive Mechanismen ausgelöst werden. Darüber hinaus zeigte diese Studie, dass die Inkubationszeit zusammen mit der Konzentration und dem Zustand des Peptids (disaggregiert versus aggregiert zu Fibrillen) ein entscheidender Faktor für die Aβ Peptid-Toxizität ist. Eine hohe Konzentration (100 µM) des fibrillären Peptids (induziert durch Lagerung bei 37 °C über Nacht) führte zum Zelltod von SH-SY5Y Zellen nach einem Tag. Allerdings war die Wirkung des disaggregierten Aß Peptids (100 μM) nach 3 Tagen Inkubation stärker ausgeprägt als bei fibrillärem Peptid. Aβ Peptid Behandlung verringerte den Prozentsatz von apoptotischen und nekrotischen Zellen (2,5-fach) in bestrahlten Zellen nach 1 Tag. Nach 3 Tagen wurde jedoch ein ~2,8-facher Anstieg von apoptotischen und nekrotischen Zellen im Vergleich zu unbestrahlten Kontrollzellen beobachtet. Der beobachtete Weg von Aβ Peptid zu zellulären Organellen und die induzierten Veränderungen der Zellphysiologie, die vom Oxidationsstress abhängen, könnten für die AD Pathologie von Bedeutung sein. Die erhaltenen Ergebnisse fordern den Einsatz von mehr physiologischen 5% O2 anstatt der nicht-physiologischen 21% O2 für die Kultivierung von SH-SY5Y Zellen, insbesondere bei Studien ihrer Reaktion auf Stress. Dies ist die Zuverlässigkeit der experimentellen Aussagen entscheidend. Zellen, die bei 5% O2 kultiviert wurden, zeigten eine höhere ATP Konzentration (~1,3-fach), eine niedrigere ROS Menge (1,5-fach), eine geringere Proteincarbonylierung (2,5-fach) und weniger mtDNA Deletion (2-fach) im Vergleich zu Zellen bei 21% O2.Deutsch
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