Hans Georg Kuhn

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain-derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfa upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Expression of ezrin radixin moesin proteins in the adult subventricular zone and the rostral migratory stream

Continuous proliferation occurs in the adult subventricular zone (SVZ) of the lateral ventricles throughout life. In the SVZ, progenitor cells differentiate into neuroblasts, which migrate tangentially along the rostral migratory stream (RMS) to reach their final destination in the olfactory bulb. These progenitor cells mature and integrate into the existing neural network of the olfactory bulb. Long distance migration of neuroblasts in the RMS requires a highly dynamic cytoskeleton with the ability to respond to surrounding stimuli. Radixin is a member of the ERM (Ezrin, Radixin, Moesin) family, which connect the actin cytoskeleton to the extracellular matrix through transmembrane proteins. The membrane-cytoskeleton linker proteins of the ERM family may regulate cellular events with a high demand on cytoskeleton plasticity, such as cell motility. Recently, specific expression of the ERM protein ezrin was shown in the RMS. Radixin however has not been characterized in this region. Here we used immunohistochemistry and confocal microscopy to examine the expression of radixin in the different cell types of the adult subventricular zone niche and in the RMS. Our findings indicate that radixin is strongly expressed in neuroblasts of the adult RMS and subventricular zone, and also in Olig2-positive cells. We also demonstrate the presence of radixin in the cerebral cortex, striatum, cerebellum, thalamus, hippocampus as well as the granular and periglomerular layers of the olfactory bulb. Our studies also reveal the localization of radixin in neurosphere culture studies and we reveal the specificity of our labeling using Western blotting. The expression pattern demonstrated here suggests a role for radixin in neuronal migration and differentiation in the adult RMS. Understanding how adult neuronal migration is regulated is of importance for the development of new therapeutic interventions using endogenous repair for neurodegenerative diseases.

Analysis of adult neurogenesis: evidence for a prominent "non-neurogenic" DCX-protein pool in rodent brain.

Here, we have developed a highly sensitive immunoassay for Dcx to characterize expression in brain and cerebrospinal fluid (CSF) of rodents. We demonstrate that Dcx is widely expressed during development in various brain regions and as well can be detected in cerebrospinal fluid of rats (up to 30 days postnatal). While Dcx protein level decline in adulthood and were detectable in neurogenic regions of the adult rodent brain, similar levels were also detectable in brain regions expected to bear no neurogenesis including the cerebral cortex and CA1/CA3 enriched hippocampus. We monitored DCX protein levels after paradigms to increase or severely decrease adult hippocampal neurogenesis, namely physical activity and cranial radiation, respectively. In both paradigms, Dcx protein- and mRNA-levels clearly reflected changes in neurogenesis in the hippocampus. However, basal Dcx-levels are unaffected in non-neurogenic regions (e.g. CA1/CA3 enriched hippocampus, cortex). These data suggest that there is a substantial “non-neurogenic” pool of Dcx- protein, whose regulation can be uncoupled from adult neurogenesis suggesting caution for the interpretation of such studies.

Characterization of epidermal growth factor-induced dysplasia in the adult rat subventricular zone.

Epidermal growth factor (EGF) is a mitogen widely used when culturing adult neural stem cells in vitro. Although proliferative effects can also be observed in vivo, intracerebroventricular infusion of EGF has been found to counteract neuronal determination and promote glial differentiation instead. However, EGF receptor activation has different effects on the subventricular zone (SVZ) in mice and rats, possibly because of species differences in SVZ cell composition. Specifically in the rat, EGF stimulation of the SVZ induces the formation of hyperplastic polyps. The present study aims at molecular and morphological characterization of these subventricular polyps. Using immunohistochemistry, electron microscopy, and gene expression analysis, we demonstrate in hyperplastic EGF-induced polyps an upregulation in protein expression of Sox2, Olig2, GFAP, nestin, and vimentin. We found polyp-specific dysplastic changes in the form of coexpression of Sox2 and Olig2. This highly proliferative, Sox2/Olig2 coexpressing dysplastic cell type is >10-fold enriched in the hyperplastic polyps compared with control SVZ and most likely causes the polyp formation. Unique ultrastructural features of the polyps include a lack of ependymal cell lining as well as a large number of cells with large, light, ovoid nuclei and a cytoplasm with abundant ribosomes, whereas other polyp cells contain invaginated nuclei but fewer ribosomes. EGF also induced changes in the expression of Id genes Id1, Id2, and Id4 in the SVZ. Taken together, we here demonstrate dysplastic, structural, and phenotypical changes in the rat SVZ following EGF stimulation, which are specific to hyperplastic polyps.

Radixin inhibition decreases adult neural progenitor cell migration and proliferation in vitro and in vivo

Neuronal progenitors capable of long distance migration are produced throughout life in the subventricular zone (SVZ). Migration from the SVZ is carried out along a well-defined pathway called the rostral migratory stream (RMS). Our recent finding of the specific expression of the cytoskeleton linker protein radixin in neuroblasts suggests a functional role for radixin in RMS migration. The ezrin-radixin-moesin (ERM) family of proteins is capable of regulating migration through interaction with the actin cytoskeleton and transmembrane proteins. The ERM proteins are differentially expressed in the RMS with radixin and moesin localized to neuroblasts, and ezrin expression confined to astrocytes of the glial tubes. Here, we inhibited radixin function using the quinocarmycin analog DX52-1 which resulted in reduced neuroblast migration in vitro, while glial migration remained unaltered. Furthermore, the morphology of neuroblasts was distorted resulting in a rounded shape with no or short polysialylated neural cell adhesion molecule positive processes. Intracerebroventricular infusion of the radixin inhibitor resulted in accumulation of neuroblasts in the anterior SVZ. Neuroblast chains were short and intermittently interrupted in the SVZ and considerably disorganized in the RMS. Moreover, we studied the proliferation activity in the RMS after radixin inhibition, since concentrated radixin expression has been demonstrated in the cleavage furrow of dividing cells, which indicates a role of radixin in cell division. Radixin inhibition decreased neuroblast proliferation, whereas the proliferation of other cells in the RMS was not affected. Our results demonstrate a significant role for radixin in neuroblast proliferation and migration.

EGF-Induced Expansion of Migratory Cells in the Rostral Migratory Stream

The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ) of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS) is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF). In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin+/Olig2+ cells in the RMS. Negative for NG2 and CNPase, these radixin+/Olig2+ cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2+ population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin+/Olig2+ progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.

Caspase inhibition impaired the neural stem/progenitor cell response after cortical ischemia in mice

Cortical ischemia induces proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) and provokes migration of these cells toward the injured area. Despite sustained migration of NSPCs for an extended period of time after injury, they do not appear to survive. Here, we hypothesized that the anti-apoptotic broad-spectrum caspase inhibitor Q-VD-OPh would increase NSPC survival in the injured cortex. However, contrary to our expectations, caspase inhibition did not promote NSPC survival and cortical neurogenesis. On the contrary, it abolished ischemia-induced proliferation and decreased the number of migrating neuroblasts in the injured cortex. Moreover, caspase inhibition decreased the levels of the chemoattractant chemokine CCL2 (MCP-1) and the pro-inflammatory cytokine IL-1β. We hence for the first time show that caspase inhibition abrogates the response of NSPCs to an ischemic injury, presumably by inhibiting the production of pro-inflammatory factors. Thus, caution is warranted if anti-apoptotic strategies are applied for neuroprotection.

Constitutive PGC-1α Overexpression in Skeletal Muscle Does Not Improve Morphological Outcome in Mouse Models of Brain Irradiation or Cortical Stroke

Physical exercise can improve morphological outcomes after ischemic stroke and ameliorate irradiation-induced reduction of hippocampal neurogenesis in rodents, but the mechanisms underlying these effects remain largely unknown. The transcription factor peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is considered to be one of the central factors responsible for exercise-induced benefits in skeletal muscle, including the release of neurotrophic factors into the circulation. In order to test if PGC-1α overexpression in skeletal muscle could simulate the exercise-induced effects on recovery after cranial irradiation and stroke, we used male adult transgenic mice overexpressing murine PGC-1α under the control of muscle creatinine kinase promoter and subjected them to either whole brain irradiation at a dose of 4u202fGy or photothrombotic stroke to the sensory motor cortex. Muscular PGC-1α overexpression did not ameliorate irradiation-induced reduction of newborn BrdU-labeled cells in the dentate gyrus, immature neurons, or newborn mature neurons. In the stroke model, muscular overexpression of PGC-1α resulted in an increased infarct size without any changes in microglia activation or reactive astrocytosis. No difference could be detected in the number of migrating neural progenitor cells from the subventricular zone to the lesioned neocortex or in vascular density of the contralateral neocortex in comparison to wildtype animals. We conclude that forced muscular overexpression of PGC-1α does not have a beneficial effect on hippocampal neurogenesis after irradiation, but rather a detrimental effect on the infarct volume after stroke in mice. This suggests that artificial muscle activation through the PGC-1α pathway is not sufficient to mimic exercise-induced recovery after cranial irradiation and stroke.

Grafting Neural Stem and Progenitor Cells Into the Hippocampus of Juvenile, Irradiated Mice Normalizes Behavior Deficits

The pool of neural stem and progenitor cells (NSPCs) in the dentate gyrus of the hippocampus is reduced by ionizing radiation. This explains, at least partly, the learning deficits observed in patients after radiotherapy, particularly in pediatric cases. An 8 Gy single irradiation dose was delivered to the whole brains of postnatal day 9 (P9) C57BL/6 mice, and BrdU-labeled, syngeneic NSPCs (1.0 × 105 cells/injection) were grafted into each hippocampus on P21. Three months later, behavior tests were performed. Irradiation impaired novelty-induced exploration, place learning, reversal learning, and sugar preference, and it altered the movement pattern. Grafting of NSPCs ameliorated or even normalized the observed deficits. Less than 4% of grafted cells survived and were found in the dentate gyrus 5 months later. The irradiation-induced loss of endogenous, undifferentiated NSPCs in the dentate gyrus was completely restored by grafted NSPCs in the dorsal, but not the ventral, blade. The grafted NSPCs did not exert appreciable effects on the endogenous NSPCs; however, more than half of the grafted NSPCs differentiated. These results point to novel strategies aimed at ameliorating the debilitating late effects of cranial radiotherapy, particularly in children.