Heinrich F. Bürgers

Matrix metalloproteinase-9 mediates hypoxia-induced vascular leakage in the brain via tight junction rearrangement

Blood–brain barrier (BBB) disruption, resulting from loss of tight junctions (TJ) and activation of matrix metalloproteinases (MMPs), is associated with edema formation in ischemic stroke. Cerebral edema develops in a phasic manner and consists of both vasogenic and cytotoxic components. Although it is contingent on several independent mechanisms, involving hypoxic and inflammatory responses, the single effect of prolonged hypoxia on BBB integrity in vivo was not addressed so far. Exposing mice to normobaric hypoxia (8% oxygen for 48 h) led to a significant increase in vascular permeability associated with diminished expression of the TJ protein occludin. Immunofluorescence studies revealed that hypoxia resulted in disrupted continuity of occludin and zonula occludens-1 (Zo-1) staining with significant gap formation. Hypoxia increased gelatinolytic activity specifically in vascular structures and gel zymography identified MMP-9 as enzymatic source. Treatment with an MMP inhibitor reduced vascular leakage and attenuated disorganization of TJ. Inhibition of vascular endothelial growth factor (VEGF) attenuated vascular leakage and MMP-9 activation induced by hypoxia. In conclusion, our data suggest that hypoxia-induced edema formation is mediated by MMP-9-dependent TJ rearrangement by a mechanism involving VEGF. Therefore, inhibition of MMP-9 might provide the basis for therapeutic strategies to treat brain edema.

Expression of Hemoglobin in Rodent Neurons

Hemoglobin is the major protein in red blood cells and transports oxygen from the lungs to oxygen-demanding tissues, like the brain. Mechanisms that facilitate the uptake of oxygen in the vertebrate brain are unknown. In invertebrates, neuronal hemoglobin serves as intracellular storage molecule for oxygen. Here, we show by immunohistochemistry that hemoglobin is specifically expressed in neurons of the cortex, hippocampus, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal hemoglobin distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels. Probing for low oxygen levels in the tissue, we provide evidence that hemoglobin α-positive cells in direct neighborhood with hemoglobin α-negative cells display a better oxygenation than their neighbors and can be sharply distinguished from those. Neuronal hemoglobin expression is upregulated by injection or transgenic overexpression of erythropoietin and is accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions. Thus we provide a novel mechanism for the neuroprotective actions of erythropoietin under ischemic—hypoxic conditions. We propose that neuronal hemoglobin expression is connected to facilitated oxygen uptake in neurons, and hemoglobin might serve as oxygen capacitator molecule.

Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression

In the brain, glucose is transported by GLUT1 across the blood–brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5‐fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb

BackgroundNeural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time.ResultsWe found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures.ConclusionIn this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

Lithium Promotes Adult Neural Progenitor Differentiation Via GSK3β- Dependent Signaling Pathways

Lithium is one of the standard drugs in the treatment of bipolar disorder, although its molecular modes of ac- tion are not well understood. It is a potent inhibitor of the multifunctional enzyme Glycogen Synthase Kinase 3s (GSK3s), which also plays a central role in neurogenesis via the developmental Wnt signaling pathway. In the present study, we analyzed the influence of lithium on GSK3s signaling in adult neural progenitor cells (NPCs) from the rat sub- ventricular zone. Protein expression patterns of NPCs cultured in the presence of 20 mM lithium chloride were compared to those of untreated cells. A proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry showed changes in several GSK3s-related proteins. We demonstrate the inhibition of GSK3s by stabilization and nuclear transfer of its downstream target, s-catenin. Moreover, the phosphorylated (=inhibited) GSK3s protein was strongly enri- ched in the nuclear fraction of lithium-treated cells. The fraction of cells differentiated into astroglia increased moderately, and the fraction of cells differentiated into neurons increased strongly, as shown by immunostaining. In conclusion, li- thium promotes NPC differentiation, mainly to neurons, via GSK3s-related pathways.