Johan Bengzon

Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.

Increased neurogenesis in a model of electroconvulsive therapy

BACKGROUND Electroconvulsive therapy (ECT) is a widely used and efficient treatment modality in psychiatry, although the basis for its therapeutic effect is still unknown. Past research has shown seizure activity to be a regulator of neurogenesis in the adult brain. This study examines the effect of a single and multiple electroconvulsive seizures on neurogenesis in the rat dentate gyrus. METHODS Rats were given either a single or a series of 10 electroconvulsive seizures. At different times after the seizures, a marker of proliferating cells, Bromodeoxyuridine (BrdU), was administered to the animals. Subsequently, newborn cells positive for BrdU were counted in the dentate gyrus. Double staining with a neuron-specific marker indicated that the newborn cells displayed a neuronal phenotype. RESULTS A single electroconvulsive seizure significantly increased the number of new born cells in the dentate gyrus. These cells survived for at least 3 months. A series of seizures further increased neurogenesis, indicating a dose-dependent mechanism. CONCLUSIONS We propose that generation of new neurons in the hippocampus may be an important neurobiologic element underlying the clinical effects of electroconvulsive seizures.

Neurotrophins and brain insults

Epileptic, hypoglycaemic, ischaemic and traumatic insults to the brain induce marked changes of gene expression for the neurotrophins, nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, and their high-affinity receptors, TrkB and TrkC, in cortical and hippocampal neurones. Release of glutamate and influx of Ca2+ are the most important triggering factors. The major hypotheses for the functional effects of the insult-induced neurotrophin changes are protection against neuronal damage and stimulation of sprouting and synaptic reorganization. More insight into the regulation and role of the neurotrophins after brain insults should increase our understanding of pathophysiological mechanisms in, for example, epileptogenesis and cell death, and could lead to new therapeutic strategies.

Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development

Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.

Increased production of the TrkB protein tyrosine kinase receptor after brain insults

The protein-tyrosine kinases Trk, TrkB, and TrkC are signal-transducing receptors for a family of neurotrophic factors known as the neurotrophins. Here we show that seizures induced by hippocampal kindling lead to a rapid, transient increase of trkB mRNA and protein in the hippocampus. TrkB is a component of a high affinity receptor for brain-derived neurotrophic factor (BDNF). No change was detected in mRNAs for Trk or TrkC, components of the high affinity nerve growth factor or neurotrophin-3 receptors, respectively. trkB mRNA was also transiently increased in the dentate gyrus following cerebral ischemia and hypoglycemic coma; these treatments had no effect on trk and trkC mRNAs. The increase in trkB mRNA and protein showed the same time course and distribution as the increase in BDNF mRNA. These data suggest that BDNF and its receptor may play a local role within the hippocampus in kindling-associated neural plasticity and in neuronal protection following epileptic, ischemic, and hypoglycemic insults.

Bone Marrow Multipotent Mesenchymal Stroma Cells Act as Pericyte-like Migratory Vehicles in Experimental Gliomas.

Bone marrow-derived multipotent mesenchymal stroma cells (MSCs) have emerged as cellular vectors for gene therapy of solid cancers. We implanted enhanced green fluorescent protein-expressing rat MSCs directly into rat malignant gliomas to address their migratory capacity, phenotype, and effects on tumor neovascularization and animal survival. A single intratumoral injection of MSCs infiltrated the majority of invasive glioma extensions (72 +/- 14%) and a substantial fraction of distant tumor microsatellites (32 +/- 6%). MSC migration was highly specific for tumor tissue. Grafted MSCs integrated into tumor vessel walls and expressed pericyte markers alpha-smooth muscle actin, neuron-glia 2, and platelet-derived growth factor receptor-beta but not endothelial cell markers. The pericyte marker expression profile and perivascular location of grafted MSCs indicate that these cells act as pericytes within tumors. MSC grafting did not influence tumor microvessel density or survival of tumor-bearing animals. The antiangiogenic drug Sunitinib markedly reduced the numbers of grafted MSCs migrating within tumors. We found no MSCs within gliomas following intravenous (i.v.) injections. Thus, MSCs should be administered by intratumoral implantations rather than by i.v. injections. Intratumorally grafted pericyte-like MSCs might represent a particularly well-suited vector system for delivering molecules to affect tumor angiogenesis and for targeting cancer stem cells within the perivascular niche.

CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells

Human brain tumor stem cells have been enriched using antibodies against the surface protein CD133. An antibody recognizing CD133 also served to isolate normal neural stem cells from fetal human brain, suggesting a possible lineage relationship between normal neural and brain tumor stem cells. Whether CD133-positive brain tumor stem cells can be derived from CD133-positive neural stem or progenitor cells still requires direct experimental evidence, and an important step toward such investigations is the identification and characterization of normal CD133-presenting cells in neurogenic regions of the embryonic and adult brain. Here, we present evidence that CD133 is a marker for embryonic neural stem cells, an intermediate radial glial/ependymal cell type in the early postnatal stage, and for ependymal cells in the adult brain, but not for neurogenic astrocytes in the adult subventricular zone. Our findings suggest two principal possibilities for the origin of brain tumor stem cells: a derivation from CD133-expressing cells, which are normally not present in the adult brain (embryonic neural stem cells and an early postnatal intermediate radial glial/ependymal cell type), or from CD133-positive ependymal cells in the adult brain, which are, however, generally regarded as postmitotic. Alternatively, brain tumor stem cells could be derived from proliferative but CD133-negative neurogenic astrocytes in the adult brain. In the latter case, brain tumor development would involve the production of CD133.

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2–mediated heparin-binding EGF signaling in endothelial cells

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein–coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2–mediated activation of hypoxic ECs. We show that PAR-2–dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2–dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa–dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2–mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

Electroconvulsive seizures increase hippocampal neurogenesis after chronic corticosterone treatment.

Major depression is often associated with elevated glucocorticoid levels. High levels of glucocorticoids reduce neurogenesis in the adult rat hippocampus. Electroconvulsive seizures (ECS) can enhance neurogenesis, and we investigated the effects of ECS in rats where glucocorticoid levels were elevated in order to mimic conditions seen in depression. Rats given injections of corticosterone or vehicle for 21 days were at the end of this period treated with either a single or five daily ECSs. Proliferating cells were labelled with bromodeoxyuridine (BrdU). After 3 weeks, BrdU‐positive cells in the dentate gyrus were quantified and analyzed for co‐labelling with the neuronal marker neuron‐specific nuclear protein (NeuN). In corticosterone‐treated rats, neurogenesis was decreased by 75%. This was counteracted by a single ECS. Multiple ECS further increased neurogenesis and no significant differences in BrdU/NeuN positive cells were detected between corticosterone‐ and vehicle‐treated rats given five ECS. Approximately 80% of the cells within the granule cell layer and 10% of the hilar cells were double‐labelled with BrdU and NeuN.

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.