Dr.rer.nat Stefan Fischer

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Zeitschriftenartikel
  • Melanie Kappelmann-Fenzl
  • S. Schmidt
  • Stefan Fischer
  • R. Schmid
  • L. Lämmerhirt
  • L. Fischer
  • S. Schrüfer
  • I. Thievessen
  • D. Schubert
  • A. Matthies
  • Rainer Detsch
  • Aldo B.
  • A. Arkudas
  • A. Kengelbach-Weigand
  • A. Bosserhoff
Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels, vol. 13, pg. 4111.

In: Cancers

  • 2021

DOI: 10.3390/cancers13164111

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.
  • Angewandte Informatik
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  • DIGITAL
Zeitschriftenartikel
  • N. Rachinger
  • Stefan Fischer
  • I. Böhme
  • L. Linck-Paulus
  • S. Kuphal
  • Melanie Kappelmann-Fenzl
  • A. Bosserhoff
Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR, vol. 22, pg. 9349.

In: International Journal of Molecular Sciences

  • 2021

DOI: 10.3390/ijms22179349

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.
  • Angewandte Informatik
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Zeitschriftenartikel
  • J. Groh
  • H. Friedman
  • N. Orel
  • C. Ip
  • Stefan Fischer
  • I. Spahn
  • E. Schäffner
  • M. Hörner
  • D. Stadler
  • M. Buttmann
  • C. Varallyay
  • L. Solymosi
  • M. Sendtner
  • A. Peterson
  • R. Martini
Pathogenic inflammation in the CNS of mice carrying human PLP1 mutations, vol. 25, pg. 4686-4702.

In: Human molecular genetics

  • 2016

DOI: 10.1093/hmg/ddw296

Progressive forms of multiple sclerosis lead to chronic disability, substantial decline in quality of life and reduced longevity. It is often suggested that they occur independently of inflammation. Here we investigated the disease progression in mouse models carrying PLP1 point mutations previously found in patients displaying clinical features of multiple sclerosis. These mouse models show loss-of-function of PLP1 associated with neuroinflammation; the latter leading to clinically relevant axonal degeneration, neuronal loss and brain atrophy as demonstrated by inactivation of the recombination activating gene 1. Moreover, these pathological hallmarks were substantially amplified when we attenuated immune regulation by inactivation of the programmed cell death-1 gene. Our observations support the view that primary oligodendroglial abnormalities can evoke pathogenically relevant neuroinflammation that drives neurodegeneration, as observed in some forms of multiple sclerosis but also in other, genetically-mediated neurodegenerative disorders of the human nervous system. As many potent immunomodulatory drugs have emerged during the last years, it is tempting to consider immunomodulation as a treatment option not only for multiple sclerosis, but also for so far non-treatable, genetically-mediated disorders of the nervous system accompanied by pathogenic neuroinflammation.
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Zeitschriftenartikel
  • F. Koch-Nolte
  • Stefan Fischer
  • F. Haag
  • M. Ziegler
Compartmentation of NAD+-dependent signalling, vol. 585, pg. 1651-6.

In: FEBS letters

  • 2011

DOI: 10.1016/j.febslet.2011.03.045

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.
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Zeitschriftenartikel
  • B. Kohl
  • Stefan Fischer
  • J. Groh
  • C. Wessig
  • R. Martini
MCP-1/CCL2 modifies axon properties in a PMP22-overexpressing mouse model for Charcot-Marie-tooth 1A neuropathy, vol. 176, pg. 1390-9.

In: The American journal of pathology

  • 2010

DOI: 10.2353/ajpath.2010.090694

Charcot-Marie-Tooth 1A (CMT1A) neuropathy, the most common inherited peripheral neuropathy, is primarily caused by a gene duplication for the peripheral myelin protein-22 (PMP22). In an accordant mouse model, we investigated the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) as a regulator of nerve macrophages and neural damage including axonopathy and demyelination. By generating PMP22tg mice with reduced levels or lack of MCP-1/CCL2, we found that MCP-1/CCL2 is involved in the increase of macrophages in mutant nerves. PMP22tg mice with wild-type levels of MCP-1/CCL2 showed strong macrophage increase in the diseased nerves, whereas either 50% reduction or total absence of MCP-1/CCL2 led to a moderate or a strong reduction of nerve macrophages, respectively. Interestingly, MCP-1/CCL2 expression level and macrophage numbers were correlated with features indicative of axon damage, such as maldistribution of K+ channels, reduced compound muscle action potentials, and muscle weakness. Demyelinating features, however, were most highly reduced when MCP-1/CCL2 was diminished by 50%, whereas complete lack of MCP-1/CCL2 showed an intermediate demyelinating phenotype. We also identified the MEK1/2-ERK1/2-pathway as being involved in MCP-1/CCL2 expression in the Schwann cells of the CMT1A model. Our data show that, in a CMT1A model, MCP-1/CCL2 activates nerve macrophages, mediates both axon damage and demyelination, and may thus be a promising target for therapeutic approaches.
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Zeitschriftenartikel
  • J. Groh
  • K. Heinl
  • B. Kohl
  • C. Wessig
  • J. Greeske
  • Stefan Fischer
  • R. Martini
Attenuation of MCP-1/CCL2 expression ameliorates neuropathy in a mouse model for Charcot-Marie-Tooth 1X, vol. 19, pg. 3530-43.

In: Human molecular genetics

  • 2010

DOI: 10.1093/hmg/ddq269

The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been previously shown to be an important mediator of macrophage-related neural damage in models of two distinct inherited neuropathies, Charcot-Marie-Tooth (CMT) 1A and 1B. In mice deficient in the gap junction protein connexin 32 (Cx32def), an established model for the X-chromosome-linked dominant form of CMT (CMT1X), we investigated the role of the chemokine in macrophage immigration and neural damage by crossbreeding the Cx32def mice with MCP-1 knockout mutants. In Cx32def mutants typically expressing increased levels of MCP-1, macrophage numbers were strongly elevated, caused by an MCP-1-mediated influx of haematogenous macrophages. Curiously, the complete genetic deletion of MCP-1 did not cause reduced macrophage numbers in the nerves due to compensatory proliferation of resident macrophages. In contrast, and as already seen in other CMT models, heterozygous deletion of MCP-1 led to reduced numbers of phagocytosing macrophages and an alleviation of demyelination. Whereas alleviated demyelination was transient, axonal damage was persistently improved and even robust axonal sprouting was detectable at 12 months. Other axon-related features were alleviated electrophysiological parameters, reduced muscle denervation and atrophy, and increased muscle strength. Similar to models for CMT1A and CMT1B, we identified MEK-ERK signalling as mediating MCP-1 expression in Cx32-deficient Schwann cells. Blocking this pathway by the inhibitor CI-1040 caused reduced MCP-1 expression, attenuation of macrophage increase and amelioration of myelin- and axon-related alterations. Thus, attenuation of MCP-1 upregulation by inhibiting ERK phosphorylation might be a promising approach to treat CMT1X and other so far untreatable inherited peripheral neuropathies in humans.
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Zeitschriftenartikel
  • S. Meuth
  • C. Kleinschnitz
  • T. Broicher
  • M. Austinat
  • S. Braeuninger
  • S. Bittner
  • Stefan Fischer
  • D. Bayliss
  • T. Budde
  • G. Stoll
  • H. Wiendl
The neuroprotective impact of the leak potassium channel TASK1 on stroke development in mice, vol. 33, pg. 1-11.

In: Neurobiology of disease

  • 2009

DOI: 10.1016/j.nbd.2008.09.006

Oxygen depletion (O(2)) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42+/-2%) and O(2) deprivation (36+/-5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24 h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1(-/-) mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.
  • Angewandte Informatik
  • GESUND
Zeitschriftenartikel
  • R. Martini
  • Stefan Fischer
  • R. López-Vales
  • S. David
Interactions between Schwann cells and macrophages in injury and inherited demyelinating disease, vol. 56, pg. 1566-77.

In: Glia

  • 2008

DOI: 10.1002/glia.20766

In this article we first discuss the factors that regulate macrophage recruitment, activation, and myelin phagocytosis during Wallerian degeneration and some of the factors involved in the termination of inflammation at the end of the period of Wallerian degeneration after peripheral nerve injuries. In particular, we deal with the early events that trigger chemokine and cytokine expression; the role of phospholipase A(2) in initiating the breakdown of compact myelin, and chemokine, cytokine expression; and the role of MCP-1, MIP-1alpha, and IL-1beta in macrophage recruitment and myelin phagocytosis. We also discuss how inflammation may be switched off and the recently identified role of the Nogo receptor on activated macrophages in the clearance of these cells from the injured nerve. In the second half of the article we focus on the role of certain Schwann cell borne cytokines and chemokines, such as M-CSF and MCP-1 as well as intracellular signaling that regulate their expression in animal models of inherited demyelinating disease. Additionally, we present the preservation of sensory nerves fibers from macrophage attack in these animal models as a challenging paradigm for the development of putative treatment approaches. Finally, we also discuss the similarities and differences in these Schwann cell-macrophage responses in injury-induced Wallerian degeneration and inherited demyelinating diseases. Knowledge of the molecular mechanisms underlying Schwann cell-macrophage interaction under pathological conditions is an important prerequisite to develop effective treatment strategies for various peripheral nerve disorders.
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Zeitschriftenartikel
  • Stefan Fischer
  • C. Kleinschnitz
  • M. Müller
  • I. Kobsar
  • C. Ip
  • B. Rollins
  • R. Martini
Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy, vol. 37, pg. 359-66.

In: Molecular and cellular neurosciences

  • 2008

DOI: 10.1016/j.mcn.2007.10.012

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.
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Zeitschriftenartikel
  • Stefan Fischer
  • A. Weishaupt
  • J. Troppmair
  • R. Martini
Increase of MCP-1 (CCL2) in myelin mutant Schwann cells is mediated by MEK-ERK signaling pathway, vol. 56, pg. 836-43.

In: Glia

  • 2008

DOI: 10.1002/glia.20657

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/-), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/- mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/- mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans.
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Zeitschriftenartikel
  • C. Ip
  • A. Kroner
  • M. Bendszus
  • C. Leder
  • I. Kobsar
  • Stefan Fischer
  • H. Wiendl
  • K. Nave
  • R. Martini
Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes, vol. 26, pg. 8206-16.

In: The Journal of Neuroscience: An Official Journal Society for Neuroscience

  • 2006

DOI: 10.1523/jneurosci.1921-06.2006

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.
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Zeitschriftenartikel
  • C. Wang Ip
  • A. Kroner
  • Stefan Fischer
  • M. Berghoff
  • I. Kobsar
  • M. Mäurer
  • R. Martini
Role of Immune Cells in Animal Models for Inherited Peripheral Neuropathies, vol. 8, pg. 175-190.

In: NeuroMolecular Medicine

  • 2006

DOI: 10.1385/NMM:8:1:175

Mice expressing half of the normal dose of protein zero (P0+/- mice) or completely deficient gap-junction protein connexin 32 -/- mice mimic demyelinating forms of inherited neuropathies, such as Charcot-Marie-Tooth (CMT) neuropathies type 1B and CMT type 1X, respectively. In both models, an almost normal myelin formation is observed during the first months of life, followed by a slowly progressing demyelinating neuropathy. In both models, there is a substantial increase of CD8+ T-lymphocytes and macrophages within the demyelinating nerves. Recently, this has also been observed in mice mildly overexpressing human peripheral myelin protein 22 kD mimicking the most common form of CMT, CMT type 1A. In all demyelinating models, the macrophages show close contacts with intact myelin sheaths or demyelinated axons, suggesting an active role of these cells in myelin degeneration. Additionally, fibroblast-like cells contact macrophages, suggesting a functional role of fibroblast-like cells in macrophage activation. By cross-breeding P0+/- and gap-junction protein connexin 32-/- mice with immunodeficient recombination activating gene-1-deficient mutants, a substantial alleviation of the demyelinating phenotype was observed. Similarly, cross-breeding of P0+/- mice with mutants with a defect in macrophage activation led to an alleviated phenotype as well. These findings demonstrate that the immune system is involved in the pathogenesis of demyelinating neuropathies. In contrast, in P0-/- mice, which display a compromised myelin compaction and axonal loss from onset, immune cells appear to have a neuroprotective effect because cross-breeding with recombination activating gene-1 mutants leads to an aggravation of axonopathic changes. In the present review, we discuss the influence of the immune system on inherited de- and dysmyelination regarding disease mechanisms and possible clinical implications.
  • Angewandte Informatik
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Zeitschriftenartikel
  • C. Ip
  • A. Kroner
  • Stefan Fischer
  • M. Berghoff
  • I. Kobsar
  • M. Mäurer
  • R. Martini
Role of immune cells in animal models for inherited peripheral neuropathies, vol. 8, pg. 175–190.

In: NeuroMolecular Medicine

  • 2006

DOI: 10.1385/nmm:8:1-2:175

Mice expressing half of the normal dose of protein zero (P0+/- mice) or completely deficient gap-junction protein connexin 32 -/- mice mimic demyelinating forms of inherited neuropathies, such as Charcot-Marie-Tooth (CMT) neuropathies type 1B and CMT type 1X, respectively. In both models, an almost normal myelin formation is observed during the first months of life, followed by a slowly progressing demyelinating neuropathy. In both models, there is a substantial increase of CD8+ T-lymphocytes and macrophages within the demyelinating nerves. Recently, this has also been observed in mice mildly overexpressing human peripheral myelin protein 22 kD mimicking the most common form of CMT, CMT type 1A. In all demyelinating models, the macrophages show close contacts with intact myelin sheaths or demyelinated axons, suggesting an active role of these cells in myelin degeneration. Additionally, fibroblast-like cells contact macrophages, suggesting a functional role of fibroblast-like cells in macrophage activation. By cross-breeding P0+/- and gap-junction protein connexin 32-/- mice with immunodeficient recombination activating gene-1-deficient mutants, a substantial alleviation of the demyelinating phenotype was observed. Similarly, cross-breeding of P0+/- mice with mutants with a defect in macrophage activation led to an alleviated phenotype as well. These findings demonstrate that the immune system is involved in the pathogenesis of demyelinating neuropathies. In contrast, in P0-/- mice, which display a compromised myelin compaction and axonal loss from onset, immune cells appear to have a neuroprotective effect because cross-breeding with recombination activating gene-1 mutants leads to an aggravation of axonopathic changes. In the present review, we discuss the influence of the immune system on inherited de- and dysmyelination regarding disease mechanisms and possible clinical implications.
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