Publications by authors named "Christoffer Tamm"

20 Publications

  • Page 1 of 1

Mechanistic insight into neurotoxicity induced by developmental insults.

Biochem Biophys Res Commun 2017 01 3;482(3):408-418. Epub 2017 Feb 3.

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. Electronic address:

Epidemiological and/or experimental studies have shown that unfavorable prenatal environmental factors, such as stress or exposure to certain neurotoxic environmental contaminants, may have adverse consequences for neurodevelopment. Alterations in neurogenesis can have harmful effects not only for the developing nervous system, but also for the adult brain where neurogenesis is believed to play a role in learning, memory, and even in depression. Many recent advances in the understanding of the complex process of nervous system development can be integrated into the field of neurotoxicology. In the past 15 years we have been using cultured neural stem or progenitor cells to investigate the effects of neurotoxic stimuli on cell survival, proliferation and differentiation, with special focus on heritable effects. This is an overview of the work performed by our group in the attempt to elucidate the mechanisms of developmental neurotoxicity and possibly provide relevant information for the understanding of the etiopathogenesis of complex brain disorders.
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http://dx.doi.org/10.1016/j.bbrc.2016.10.087DOI Listing
January 2017

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture.

Nat Commun 2016 07 13;7:12170. Epub 2016 Jul 13.

Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, 751 23 Uppsala, Sweden.

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.
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http://dx.doi.org/10.1038/ncomms12170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947164PMC
July 2016

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.

Stem Cell Rev Rep 2016 Oct;12(5):584-591

Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23, Uppsala, Sweden.

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.
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http://dx.doi.org/10.1007/s12015-016-9673-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050252PMC
October 2016

Serum Inter-α-inhibitor activates the Yes tyrosine kinase and YAP/TEAD transcriptional complex in mouse embryonic stem cells.

J Biol Chem 2014 Nov 9;289(48):33492-502. Epub 2014 Oct 9.

From the Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75 123 Uppsala, Sweden and GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal.
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http://dx.doi.org/10.1074/jbc.M114.580076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246103PMC
November 2014

A comparative study of protocols for mouse embryonic stem cell culturing.

PLoS One 2013 10;8(12):e81156. Epub 2013 Dec 10.

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858223PMC
March 2015

MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to vascular endothelial growth factor A (VEGFA).

J Biol Chem 2013 Sep 24;288(36):25956-25963. Epub 2013 Jul 24.

From the Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Husargatan 3, P. O. Box 582, SE-751 23 Uppsala,. Electronic address:

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.
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http://dx.doi.org/10.1074/jbc.M113.484360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764800PMC
September 2013

Heparan sulfate biosynthesis enzymes in embryonic stem cell biology.

J Histochem Cytochem 2012 Dec 4;60(12):943-9. Epub 2012 Oct 4.

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can give rise to all cell types in the body. The fate of ES cells depends on the signals they receive from their surrounding environment, which either promote self-renewal or initiate differentiation. Heparan sulfate proteoglycans are macromolecules found on the cell surface and in the extracellular matrix. Acting as low-affinity receptors on the cell surface, heparan sulfate (HS) side chains modulate the functions of numerous growth factors and morphogens, having wide impact on the extracellular information received by cells. ES cells lacking HS fail to differentiate but can be induced to do so by adding heparin. ES cells defective in various components of the HS biosynthesis machinery, thus expressing differently flawed HS, exhibit lineage-specific effects. Here we discuss recent studies on the biological functions of HS in ES cell developmental processes. Since ES cells have significant potential applications in tissue/cell engineering for cell replacement therapies, understanding the functional mechanisms of HS in manipulating ES cell growth in vitro is of utmost importance, if the stem cell regenerative medicine from scientific fiction ever will be made real.
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http://dx.doi.org/10.1369/0022155412465090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527887PMC
December 2012

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors.

Exp Cell Res 2012 Feb 17;318(4):336-49. Epub 2011 Dec 17.

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.
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http://dx.doi.org/10.1016/j.yexcr.2011.12.008DOI Listing
February 2012

Regulation of mouse embryonic stem cell self-renewal by a Yes-YAP-TEAD2 signaling pathway downstream of LIF.

J Cell Sci 2011 Apr 8;124(Pt 7):1136-44. Epub 2011 Mar 8.

Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden.

The cytoplasmic tyrosine kinase Yes has previously been shown to have an important role in maintaining mouse and human embryonic stem (ES) self-renewal through an unknown pathway downstream of leukemia inhibitory factor (LIF) and one or more factors in serum. Here, we show that TEAD2 and its transcriptional co-activator, the Yes-associated protein YAP, co-operate in a signaling pathway downstream of Yes. We show that YAP, TEAD2 and Yes are highly expressed in self-renewing ES cells, are activated by LIF and serum, and are downregulated when cells are induced to differentiate. We also demonstrate that kinase-active Yes binds and phosphorylates YAP, and activates YAP-TEAD2-dependent transcription. We found that TEAD2 associates directly with the Oct-3/4 promoter. Moreover, activation of the Yes pathway induced activity of the Oct-3/4 and Nanog promoters, whereas suppression of this pathway inhibited promoter activity. Nanog, in turn, suppressed TEAD2-dependent promoter activity, whereas siRNA-mediated knockdown of Nanog induced it, suggesting a negative regulatory feedback loop. Episomal supertransfection of cells with inhibitory TEAD2-EnR induced endodermal differentiation, which suggests that this pathway is necessary for ES cell maintenance.
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http://dx.doi.org/10.1242/jcs.075796DOI Listing
April 2011

Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells.

Cell Cycle 2008 Oct 20;7(20):3225-34. Epub 2008 Oct 20.

Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, Linköping, Sweden.

One of the earliest morphological changes occurring in apoptosis is cell shrinkage associated with an increased efflux of K(+) and Cl(-) ions. Block of K(+) or Cl(-) channels prevents cell shrinkage and death. Recently, we found evidences for the activation of a voltage-dependent anion channel in the plasma membrane (pl-VDAC) of a hippocampal cell line undergoing apoptosis. Nothing is known on pl-VDAC in apoptotic cell death of neural cells at different stages of differentiation. We have addressed this issue in primary cultures of differentiated hippocampal neurons and embryonic neural stem cells (NSCs). In control hippocampal neurons, pl-VDAC is closed but acts as an NADH-ferricyanide reductase, while in apoptotic neurons, pl-VDAC is opened and the enzymatic activity is increased. Anti-VDAC antibodies block pl-VDAC and prevent apoptosis, as well as the increase in enzymatic activity. Conversely, in NSCs, pl-VDAC is scarcely seen and there is no NADH-ferricyanide reductase activity. In agreement, anti-VDAC antibodies do not affect the apoptotic process. Instead, we find activation of a Na(+) channel that has low voltage dependency, a conductance of 26 pS, and is blocked by amiloride, which also prevents apoptosis. Thus, it appears that activation of pl-VDAC during apoptosis is a critical event in differentiated neurons, but not in NSCs.
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http://dx.doi.org/10.4161/cc.7.20.6831DOI Listing
October 2008

Methylmercury inhibits differentiation of rat neural stem cells via Notch signalling.

Neuroreport 2008 Feb;19(3):339-43

Division of Toxicology and Neurotoxicology, Karolinska Institutet, Stockholm, Sweden.

The Notch receptor is essential for neural stem cell (NSC) characteristics. Relatively high concentrations (micromolar) of methylmercury (MeHg) activate Notch signalling in Drosophila cell lines; however, exposure of MeHg at such concentrations is rare, and the implications for mammalian cells are unclear. We have shown that MeHg at a nanomolar range inhibits neuronal differentiation of rodent embryonic NSCs. Here we show that low MeHg levels (2.5-10 nM) activated Notch signalling in NSCs, as assessed by the increased activity in a specific Notch-reporter assay and by the increased cleavage of the Notch intracellular domain. Importantly, pretreatment with Notch cleavage inhibitor reversed the MeHg-induced repression of neuronal differentiation, suggesting that Notch activation is involved in the inhibition of NSC differentiation by environmentally relevant levels of MeHg.
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http://dx.doi.org/10.1097/WNR.0b013e3282f50ca4DOI Listing
February 2008

Caspase-2 activation in neural stem cells undergoing oxidative stress-induced apoptosis.

Apoptosis 2008 Mar;13(3):354-63

Division of Toxicology and Neurotoxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77, Stockholm, Sweden.

Oxidative stress occurs as a consequence of disturbance in the balance between the generation of reactive oxygen species (ROS) and the antioxidant defence mechanisms. The interaction of ROS with DNA can cause single-, or double-strand breaks that subsequently can lead to the activation of p53, which is central for the regulation of cellular response, e.g. apoptosis, to a range of environmental and intracellular stresses. Previous reports have suggested a regulatory role of p53 in the early activation of caspase-2, upstream of mitochondrial apoptotic signaling. Here we show that excessive ROS formation, induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) exposure, induces apoptosis in primary cultured neural stem cells (NSCs) from cortices of E15 rat embryos. Following DMNQ exposure cells exhibited apoptotic hallmarks such as Bax oligomerization and activation, cytochrome c release, caspase activation and chromatin condensation. Additionally, we could show early p53 accumulation and a subsequent activation of caspase-2. The attenuation of caspase-2 activity with selective inhibitors could antagonize the mitochondrial signaling pathway and cell death. Overall, our results strongly suggest that DMNQ-induced oxidative stress causes p53 accumulation and consequently caspase-2 activation, which in turn initiates apoptotic cell death via the mitochondria-mediated caspase-dependent pathway in NSCs.
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http://dx.doi.org/10.1007/s10495-007-0172-7DOI Listing
March 2008

Mitochondrial-mediated apoptosis in neural stem cells exposed to manganese.

Toxicol Sci 2008 Feb 31;101(2):310-20. Epub 2007 Oct 31.

Division of Toxicology and Neurotoxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

Manganese is an essential nutrient for humans that has to be maintained at proper levels for normal brain functioning. However, manganese also acts as a toxicant to the brain, and several studies have linked exposure to excessive manganese to neurotoxicity in adults. A recent report has suggested that ingesting high doses of manganese via drinking water can impede intellectual functions in children. It is known that during development, the nervous system is particularly vulnerable to different types of injuries and toxicants. Neural stem cells (NSCs) play an essential role in both the developing nervous system and the adult brain where the capacity for self-renewal may be important. In the present study, we have used NSCs to investigate the molecular mechanisms involved in manganese developmental neurotoxicity. The results show that primary cultures of rat embryonic cortical NSCs as well as the murine-derived multipotent NSC line C17.2 undergo apoptotic cell death via a mitochondrial-mediated pathway in response to manganese. Exposed cells exhibit typical apoptotic features, such as chromatin condensation and cell shrinkage, mitochondrial cytochrome c release, activation of caspase-3, and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. In addition, our data also show that reactive oxygen species formation plays a role in the onset of manganese toxicity in NSCs.
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http://dx.doi.org/10.1093/toxsci/kfm267DOI Listing
February 2008

Mechanisms and modulation of neural cell damage induced by oxidative stress.

Physiol Behav 2007 Sep 24;92(1-2):87-92. Epub 2007 May 24.

Karolinska Institutet, Division of Toxicology and Neurotoxicology, Institute of Environmental Medicine, S-171 77 Stockholm, Sweden.

Oxidative stress has been linked to several neurodegenerative disorders characterized by neuronal death. Apoptosis and necrosis are the two major forms of cell death that have been described in the nervous system, and stimuli inducing oxidative stress can cause both types of death, depending on the intensity and the duration of the insult. In the present article, we report on a series of studies from our laboratory describing the intracellular pathways activated by oxidative stress in differentiated neurons, such as cerebellar granule cells, and neural stem cells. Using in vitro/ex vivo experimental models, we have investigated whether the susceptibility to injuries can be affected by the occurrence of potential insults taking place during development. We have found that prenatal exposure to high levels of glucocorticoids renders neural cells, including stem cells, more sensitive to oxidative stress damage. Similar effects were seen after in utero exposure to methylmercury. The analysis of behavior has proven to be a sensitive tool to detect mild alterations induced by early stimuli that increase susceptibility to oxidative stress. Our findings contribute to the understanding of how early events may have long-term consequences by modifying intracellular processes that predispose the affected cells to dysfunction, which can be unmasked or worsen by subsequent exposure to further injuries.
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http://dx.doi.org/10.1016/j.physbeh.2007.05.048DOI Listing
September 2007

Developmental exposure to methylmercury alters learning and induces depression-like behavior in male mice.

Toxicol Sci 2007 Jun 4;97(2):428-37. Epub 2007 Jan 4.

Division of Toxicology and Neurotoxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

To investigate the long-term effects of developmental exposure to methylmercury (MeHg), pregnant mice were exposed to at 0.5 mg MeHg/kg/day via drinking water from gestational day 7 until day 7 after delivery. The behavior of offspring was monitored at 5-15 and 26-36 weeks of age using an automated system (IntelliCage) designed for continuous long-term recording of the home cage behavior in social groups and complex analysis of basic activities and learning. In addition, spontaneous locomotion, motor coordination on the accelerating rotarod, spatial learning in Morris water maze, and depression-like behavior in forced swimming test were also studied. The analysis of behavior performed in the IntelliCage without social deprivation occurred to be more sensitive in detecting alterations in activity and learning paradigms. We found normal motor function but decreased exploratory activity in MeHg-exposed male mice, especially at young age. Learning disturbances observed in MeHg-exposed male animals suggest reference memory impairment. Interestingly, the forced swimming test revealed a predisposition to depressive-like behavior in the MeHg-exposed male offspring. This study provides novel evidence that the developmental exposure to MeHg can affect not only cognitive functions but also motivation-driven behaviors.
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http://dx.doi.org/10.1093/toxsci/kfl199DOI Listing
June 2007

Cell death mechanisms in AtT20 pituitary cells exposed to polychlorinated biphenyls (PCB 126 and PCB 153) and methylmercury.

Toxicol Lett 2006 Dec 22;167(3):183-90. Epub 2006 Sep 22.

Division of Toxicology and Neurotoxicology, Institute of Environmental Medicine, Karolinska Institutet, S-17177 Stockholm, Sweden.

Polychlorinated biphenyls (PCBs) are persistent food contaminants that can have adverse effects on the endocrine and nervous systems, including the pituitary. In the present study, we have investigated cell death in the AtT20 pituitary cell line after exposure to coplanar PCB 126 and non-coplanar PCB 153. In addition, co-exposure to the PCBs and another neurotoxic food contaminant, methylmercury (MeHg), was studied to test possible interactive effects. Our results show that mainly necrosis is induced after exposure to the selected toxicants. Simultaneous exposure to moderately toxic doses of PCBs and MeHg resulted in additive or slightly synergistic effects on the induction of cell death. Furthermore, our data suggest that both PCB congeners trigger cell death in AtT20 cells via activation of calcium regulated calpains and lysosomal cathepsins, possibly through disruption of mitochondrial function and intracellular calcium signaling. However, caspase-activity appears not to be critical for PCB induced cell death in these cells. Presence of reactive oxygen species (ROS) and protective effects of pre-treatment with antioxidants were only found after MeHg exposure, suggesting that oxidative stress plays a major role in MeHg but not PCB toxicity in this experimental model.
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http://dx.doi.org/10.1016/j.toxlet.2006.09.006DOI Listing
December 2006

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation.

J Neurochem 2006 Apr 8;97(1):69-78. Epub 2006 Mar 8.

Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, Stockholm, Sweden.

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.
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http://dx.doi.org/10.1111/j.1471-4159.2006.03718.xDOI Listing
April 2006

915 MHz microwaves and 50 Hz magnetic field affect chromatin conformation and 53BP1 foci in human lymphocytes from hypersensitive and healthy persons.

Bioelectromagnetics 2005 Apr;26(3):173-84

Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden.

We used exposure to microwaves from a global system for mobile communication (GSM) mobile phone (915 MHz, specific absorption rate (SAR) 37 mW/kg) and power frequency magnetic field (50 Hz, 15 muT peak value) to investigate the response of lymphocytes from healthy subjects and from persons reporting hypersensitivity to electromagnetic field (EMF). The hypersensitive and healthy donors were matched by gender and age and the data were analyzed blind to treatment condition. The changes in chromatin conformation were measured with the method of anomalous viscosity time dependencies (AVTD). 53BP1 protein, which has been shown to colocalize in foci with DNA double strand breaks (DSBs), was analyzed by immunostaining in situ. Exposure at room temperature to either 915 MHz or 50 Hz resulted in significant condensation of chromatin, shown as AVTD changes, which was similar to the effect of heat shock at 41 degrees C. No significant differences in responses between normal and hypersensitive subjects were detected. Neither 915 MHz nor 50 Hz exposure induced 53BP1 foci. On the contrary, a distinct decrease in background level of 53BP1 signaling was observed upon these exposures as well as after heat shock treatments. This decrease correlated with the AVTD data and may indicate decrease in accessibility of 53BP1 to antibodies because of stress-induced chromatin condensation. Apoptosis was determined by morphological changes and by apoptotic fragmentation of DNA as analyzed by pulsed-field gel electrophoresis (PFGE). No apoptosis was induced by exposure to 50 Hz and 915 MHz microwaves. In conclusion, 50 Hz magnetic field and 915 MHz microwaves under specified conditions of exposure induced comparable responses in lymphocytes from healthy and hypersensitive donors that were similar but not identical to stress response induced by heat shock.
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http://dx.doi.org/10.1002/bem.20103DOI Listing
April 2005

Differential regulation of the mitochondrial and death receptor pathways in neural stem cells.

Eur J Neurosci 2004 May;19(10):2613-21

Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, 71 77 Stockholm, Sweden.

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine-derived multipotent C17.2 NSCs, that cells undergo mitochondria-mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase-9 and -3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase-8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal-regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.
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http://dx.doi.org/10.1111/j.0953-816X.2004.03391.xDOI Listing
May 2004

Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3.

J Neurochem 2004 Jan;88(2):462-8

Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden.

Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 microm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 microm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.
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http://dx.doi.org/10.1046/j.1471-4159.2003.02162.xDOI Listing
January 2004