Phosphatidylinositol 3 kinase (PI3-kinase) is activated during and is required for hippocampal gl... more Phosphatidylinositol 3 kinase (PI3-kinase) is activated during and is required for hippocampal glutamate receptor-dependent long-term potentiation. It mediates the delivery of AMPA receptors to the neuronal surface. Among the downstream targets of PI3-kinase are three members of the serum- and glucocorticoid-inducible kinase family, SGK1, SGK2 and SGK3. In Xenopus oocytes expressing the AMPA subunit GluR1, we show that SGK3, and to a lesser extent SGK2, but not SGK1, increase glutamate-induced currents by increasing the abundance of GluR1 protein in the cell membrane. We further show Sgk3 mRNA expression in the hippocampus by RT-PCR and in situ hybridization. According to Western blotting, the hippocampal abundance of GluR1 is significantly lower in gene-targeted mice lacking SGK3 (Sgk3-/-) than in their wild-type littermates (Sgk3+/+). The present observations disclose a novel mechanism in the regulation of GluR1.
Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attemp... more Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3β (GSK-3β), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3β phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3β inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3β using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3β in cell death in response to excitotoxins in vivo and further confirms that GSK-3β plays a role in cell death/cell survival pathways.
In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cell... more In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.
Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine te... more Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology. Four adult warmblood horses received a unilateral injection of 10 × 10(6) autologous AT-MSCs into surgically created front-limb SDFT lesions. Administered AT-MSCs expressed lentivirally transduced reporter genes for GFP and were co-labelled with SPIO particles in three horses. The presence of AT-MSCs in SDFTs was evaluated by repeated examinations with standing low-field MRI in two horses and post-mortem in all horses with Prussian blue staining, fluorescence microscopy and with immunofluorescence and immunohistochemistry using anti-GFP antibodies at 3, 5, 7 and 9 weeks after treatment. AT-MSCs labelled with SPIO particles were detectable in treated SDFTs during each MRI in T2*- and T1-weighted sequences until the end of the observation period. Post-mortem examinations revealed that all treated tendons contained high numbers of SPIO- and GFP-labelled cells. Standing low-field MRI has the potential to track SPIO-labelled AT-MSCs successfully. Histology, fluorescence microscopy, immunofluorescence and immunohistochemistry are efficient tools to detect labelled AT-MSCs after intralesional injection into surgically created equine SDFT lesions. Intralesional injection of 10 × 10(6) AT-MSCs leads to the presence of high numbers of AT-MSCs in and around surgically created tendon lesions for up to 9 weeks. Integration of injected AT-MSCs into healing tendon tissue is an essential pathway after intralesional administration. Injection techniques have to be chosen deliberately to avoid reflux of the cell substrate injected. In vivo low-field MRI may be used as a non-invasive tool to monitor homing and engraftment of AT-MSCs in horses with tendinopathy of the SDFT.
The aim of this study was to elucidate the molecular status of single human adult germ stem cells... more The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-ce...
Immunocytochemical double labelling was used to determine the structural relationship of oxytocin... more Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.
Amyloid β peptide, a major component of Alzheimer's disease plaques, is directly toxic to various... more Amyloid β peptide, a major component of Alzheimer's disease plaques, is directly toxic to various neuronal cell lines and primary neurons in culture. The mechanism underlying Aβ neurotoxicity may include au increase in intracellular calcium and reactive oxygen species. In the present study, exposure of a mouse hippocampal cell line (HT-22) to the 25–35 peptide fragment of Aβ (10 μM) caused a rapid and sustained increase in nuclear c-Fos immunoreactivity. Inhibition of A β-mediated c-Fos activation by c-fos antisense oligodeoxynucleotides (5 μM) significantly protected against Aβ toxicity as assessed by MTT assay. The signal transduction pathway for c-fos induction remains speculative, however, there seems to be a causal relationship between c-Fos transcription factor and Aβ toxicity.
Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an ... more Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an increasing number of patients ask for reconstruction of the outer, striated urethral sphincter as therapy for urinary stress incontinence. Regenerative medicine is starting to offer solutions using stem cells as a part of oncological therapy or in reconstructive surgery. In addition to the many auspicious experimental approaches, one published study reports the effective therapeutic use of myogenic stem cells in urinary stress incontinent patients. Before this procedure is adopted into general clinical practice, further studies with validated evaluations and a sound legal basis are needed.
MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an importan... more MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells.
Here, we aimed to answer important and fundamental questions in germ cell biology with special fo... more Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.
In situ hybridization revealed that c-fos encoding transcripts occur in the median eminence of ra... more In situ hybridization revealed that c-fos encoding transcripts occur in the median eminence of rats. Osmotic stress resulted within 15 min. in additional labeling of the magnocellular nuclei, while osmotically stimulated rats that had been pretreated with colchicine failed to show c-fos hybridization in the magnocellular perikarya. Rats, pretreated with a polymerase II inhibitor, showed 15 min. after osmotic stimulation c-fos hybridization in the hypothalamic nuclei, whereas the median eminence was depleted of hybridization signal. c-fos is probably among the transcripts, stored in axons of the hypothalamo-neurohypophysial system, to be transported retrogradly upon osmotic stimulation.
Phosphatidylinositol 3 kinase (PI3-kinase) is activated during and is required for hippocampal gl... more Phosphatidylinositol 3 kinase (PI3-kinase) is activated during and is required for hippocampal glutamate receptor-dependent long-term potentiation. It mediates the delivery of AMPA receptors to the neuronal surface. Among the downstream targets of PI3-kinase are three members of the serum- and glucocorticoid-inducible kinase family, SGK1, SGK2 and SGK3. In Xenopus oocytes expressing the AMPA subunit GluR1, we show that SGK3, and to a lesser extent SGK2, but not SGK1, increase glutamate-induced currents by increasing the abundance of GluR1 protein in the cell membrane. We further show Sgk3 mRNA expression in the hippocampus by RT-PCR and in situ hybridization. According to Western blotting, the hippocampal abundance of GluR1 is significantly lower in gene-targeted mice lacking SGK3 (Sgk3-/-) than in their wild-type littermates (Sgk3+/+). The present observations disclose a novel mechanism in the regulation of GluR1.
Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attemp... more Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3β (GSK-3β), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3β phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3β inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3β using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3β in cell death in response to excitotoxins in vivo and further confirms that GSK-3β plays a role in cell death/cell survival pathways.
In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cell... more In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.
Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine te... more Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology. Four adult warmblood horses received a unilateral injection of 10 × 10(6) autologous AT-MSCs into surgically created front-limb SDFT lesions. Administered AT-MSCs expressed lentivirally transduced reporter genes for GFP and were co-labelled with SPIO particles in three horses. The presence of AT-MSCs in SDFTs was evaluated by repeated examinations with standing low-field MRI in two horses and post-mortem in all horses with Prussian blue staining, fluorescence microscopy and with immunofluorescence and immunohistochemistry using anti-GFP antibodies at 3, 5, 7 and 9 weeks after treatment. AT-MSCs labelled with SPIO particles were detectable in treated SDFTs during each MRI in T2*- and T1-weighted sequences until the end of the observation period. Post-mortem examinations revealed that all treated tendons contained high numbers of SPIO- and GFP-labelled cells. Standing low-field MRI has the potential to track SPIO-labelled AT-MSCs successfully. Histology, fluorescence microscopy, immunofluorescence and immunohistochemistry are efficient tools to detect labelled AT-MSCs after intralesional injection into surgically created equine SDFT lesions. Intralesional injection of 10 × 10(6) AT-MSCs leads to the presence of high numbers of AT-MSCs in and around surgically created tendon lesions for up to 9 weeks. Integration of injected AT-MSCs into healing tendon tissue is an essential pathway after intralesional administration. Injection techniques have to be chosen deliberately to avoid reflux of the cell substrate injected. In vivo low-field MRI may be used as a non-invasive tool to monitor homing and engraftment of AT-MSCs in horses with tendinopathy of the SDFT.
The aim of this study was to elucidate the molecular status of single human adult germ stem cells... more The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-ce...
Immunocytochemical double labelling was used to determine the structural relationship of oxytocin... more Immunocytochemical double labelling was used to determine the structural relationship of oxytocin (OT) and tyrosine hydroxylase (TH) containing perikarya and processes in the rat hypothalamus. Extrahypothalamic TH fibers, as well as parvocellular TH neurons were found to form contacts with OT cells. A fraction of the OT neurons contained TH immunoreactivity. It is likely that in addition to the classical mesencephalic afferences also hypothalamic interneurons and magnocellular dopaminergic neurons control the hypothalamo neurohypophysial system.
Amyloid β peptide, a major component of Alzheimer's disease plaques, is directly toxic to various... more Amyloid β peptide, a major component of Alzheimer's disease plaques, is directly toxic to various neuronal cell lines and primary neurons in culture. The mechanism underlying Aβ neurotoxicity may include au increase in intracellular calcium and reactive oxygen species. In the present study, exposure of a mouse hippocampal cell line (HT-22) to the 25–35 peptide fragment of Aβ (10 μM) caused a rapid and sustained increase in nuclear c-Fos immunoreactivity. Inhibition of A β-mediated c-Fos activation by c-fos antisense oligodeoxynucleotides (5 μM) significantly protected against Aβ toxicity as assessed by MTT assay. The signal transduction pathway for c-fos induction remains speculative, however, there seems to be a causal relationship between c-Fos transcription factor and Aβ toxicity.
Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an ... more Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an increasing number of patients ask for reconstruction of the outer, striated urethral sphincter as therapy for urinary stress incontinence. Regenerative medicine is starting to offer solutions using stem cells as a part of oncological therapy or in reconstructive surgery. In addition to the many auspicious experimental approaches, one published study reports the effective therapeutic use of myogenic stem cells in urinary stress incontinent patients. Before this procedure is adopted into general clinical practice, further studies with validated evaluations and a sound legal basis are needed.
MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an importan... more MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells.
Here, we aimed to answer important and fundamental questions in germ cell biology with special fo... more Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.
In situ hybridization revealed that c-fos encoding transcripts occur in the median eminence of ra... more In situ hybridization revealed that c-fos encoding transcripts occur in the median eminence of rats. Osmotic stress resulted within 15 min. in additional labeling of the magnocellular nuclei, while osmotically stimulated rats that had been pretreated with colchicine failed to show c-fos hybridization in the magnocellular perikarya. Rats, pretreated with a polymerase II inhibitor, showed 15 min. after osmotic stimulation c-fos hybridization in the hypothalamic nuclei, whereas the median eminence was depleted of hybridization signal. c-fos is probably among the transcripts, stored in axons of the hypothalamo-neurohypophysial system, to be transported retrogradly upon osmotic stimulation.
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Papers by Thomas Skutella