Thorsten Trapp

Monitoring of implanted stem cell migration in vivo: A highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-μm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.

Host-Dependent Tumorigenesis of Embryonic Stem Cell Transplantation in Experimental Stroke

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

Progesterone receptor-mediated effects of neuroactive steroids

Several 3 alpha-hydroxysteroids accumulate in the brain after local synthesis or after metabolization of steroids that are provided by the adrenals. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone are believed not to interact with intracellular receptors, but enhance GABA-mediated chloride currents. The present study shows that these neuroactive steroids can regulate gene expression via the progesterone receptor. The induction of DNA binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neuroactive steroids into progesterone receptor active 5 alpha-pregnane steroids. Thus, at physiological concentrations, these neuroactive steroids regulate neuronal function through their effects on both transmitter-gated ion channels and steroid receptor-regulated gene expression.

Heterodimerization between mineralocorticoid and glucocorticoid receptor: A new principle of glucocorticoid action in the CNS

In the mammalian central nervous system, responsiveness to glucocorticoids is mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These pharmacologically distinct receptors are believed to bind to common response elements as homodimers. We provide evidence that MR and GR can form a heterodimeric complex with DNA-binding and transactivation properties different from those of the respective homodimers. There was a high degree of cooperativity of MR and GR in binding to a glucocorticoid response element. Transient transfection of a neuroblastoma cell line revealed a transcriptional response pattern of coexpressed MR and GR distinct from that obtained by MR or GR alone. Our findings demonstrate that heterodimerization of MR and GR is a hitherto unrecognized principle for the transcriptional regulation of glucocorticoid-responsive genes in tissue coexpressing these receptors.

Heterodimerization between mineralocorticoid and glucocorticoid receptors increases the functional diversity of corticosteroid action

Gene regulation by steroids is mediated by the binding of the endogenous or pharmacological ligand to the corresponding nuclear receptor. Ligand-activated steroid receptors usually regulate the expression of responsive genes by binding to common response elements on DNA as homodimers. However, recent findings indicate that mineralocorticoid and glucocorticoid receptors are able to interact by forming heterodimers. In tissues coexpressing both of these corticosteroid receptors, heterodimerization between them may be a hitherto unrecognized modality for the transcriptional regulation of corticosteroid-responsive genes. In this review, Thorsten Trapp and Florian Holsboer discuss the potential impact of this heterodimerization on corticosteriod physiology and pharmacology.

The unliganded estrogen receptor (ER) transduces growth factor signals

In the absence of serum and estrogen, we show that the growth of the prolactin secreting pituitary tumour cell line, GH3 is stimulated by insulin and insulin-like growth factor-1 (IGF-1) and this response is blocked by the steroidal antiestrogens, ICI 164384 and ICI 182780. From conditioned medium (CM) experiments, growth of low density cells (10k/cm2) is increased by the addition of CM from high density cells (100k/cm2) and this growth effect is also blocked by antiestrogen. Transfection studies with a delta MTV-ERE-LUC reporter plasmid show that in the absence of estrogen and serum, both insulin and IGF-1 induce luciferase expression and this is blocked by the pure antiestrogens. No effect of these treatments was apparent when parallel experiments were conducted with a plasmid construct lacking the vitellogenin estrogen response element. From these and other data discussed in this report, we conclude that for GH3 cells, in the absence of estrogen and serum, the ER is transcriptionally activated by intracellular peptide factor pathways and by this means, acts as the key nuclear factor inducing mitogenesis in response to autocrine and exogenously added growth factors.

PROTECTION AGAINST OXIDATIVE STRESS-INDUCED NEURONAL CELL DEATH : A NOVEL ROLE FOR RU486

Free radicals and oxidative stress‐induced neuronal cell death have been implicated in a variety of neurological disorders. Therefore, neuroprotection is of primary interest in basic and preclinical neuroscience. Here it is shown that RU486 (mifepristone), a potent antagonist of progesterone and glucocorticoid receptors, protects rat primary hippocampal neurons, clonal mouse hippocampal cells and organotypic hippocampal slice cultures against oxidative stress‐induced neuronal cell death. 10‐5 M RU486 prevents intracellular peroxide accumulation and cell death induced by amyloid β protein, hydrogen peroxide and glutamate, neurotoxins that have been implicated in certain neurodegenerative disorders, including Alzheimers disease. RU486 has a significant protective effect that is independent of the presence and activation of glucocorticoid or progesterone receptors. The neuroprotective activity of this well‐studied drug may have an impact on therapeutic interventions for neurodegenerative conditions which involve peroxidation processes. such as stroke and Alzheimers disease.

Transgenic mice overexpressing XIAP in neurons show better outcome after transient cerebral ischemia

X-chromosome linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein (IAP) family and known to inhibit death of various cells under different experimental conditions. Although present in brain tissue, little is known about the physiology of the IAPs in nerve cells. Here we report on the establishment of transgenic mice with overexpression of human XIAP in brain neurons. The mice developed normally, and were more resistant to brain injury caused by transient forebrain ischemia after occlusion of the middle cerebral artery compared to control mice. The XIAP transgenic animals exhibited significantly smaller brain damage, as shown by TUNEL labelling, less reduction in brain protein synthesis, and less active caspase-3 after ischemia compared with controls. Upregulation of RhoB, which is an early indicator of neurological damage, was markedly reduced in the XIAP-overexpressing mice, which had also a better neurological outcome than control animals. This together with the increase in XIAP in normal mouse brain in regions surviving the infarct demonstrates that XIAP is an important factor promoting neuronal survival after ischemia. The results suggest that interference with the levels and the activity of XIAP in neurons may provide targets for the development of drugs limiting neuronal death after ischemia, and possibly in other brain injuries.

GTPase RhoB: An Early Predictor of Neuronal Death after Transient Focal Ischemia in Mice

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.

Hepatocyte Growth Factor/c-MET Axis-mediated Tropism of Cord Blood-derived Unrestricted Somatic Stem Cells for Neuronal Injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.