[Show abstract][Hide abstract] ABSTRACT: Adult hippocampal neurogenesis is to a large degree controlled at the level of cell survival, and a number of potential mediators of this effect have been postulated. Here, we investigated the small heat shock protein Hspb8, which, because of its pleiotropic prosurvival effects in other systems, was considered a particularly promising candidate factor. Hspb8 is, for example, found in plaques of Alzheimer disease but exerts neuroprotective effects. We found that expression of Hspb8 increased during differentiation in vitro and was particularly associated with later stages (48 –96 h) of differentiation. Gain-of-function and loss-of-function experiments supported the hypothesis that Hspb8 regulates cell survival of new neurons in vitro. In the dentate gyrus of adult mice in vivo, lentiviral overexpression of Hspb8 doubled the surviving cells and concomitantly promoted differentiation and net neurogenesis without affecting precursor cell prolifer-ation. We also discovered that the truncated form of the crystallin domain of Hspb8 was sufficient to affect cell survival and neuronal differentiation in vitro and in vivo. Precursor cell experiments in vitro revealed that Hspb8 increases the phosphorylation of Akt and suggested that the prosurvival effect can be produced by a cell-autonomous mechanism. Analysis of hippocampal Hspb8 expression in mice of 69 strains of the recombinant inbred set BXD revealed that Hspb8 is a cis-acting gene whose expression was associated with clusters of transcript enriched in genes linked to growth factor signaling and apoptosis. Our results strongly suggest that Hspb8 and its -crystallin domain might act as pleiotropic prosurvival factor in the adult hippocampus.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 03/2013; 33((13)):5785-5796. DOI:10.1523/JNEUROSCI.6452-11.2013 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The value of bioluminescence imaging (BLI) for experimental cancer models has become firmly established. We applied BLI to the GL261 glioma model in the context of dendritic cell (DC) immunotherapy. Initial validation revealed robust linear correlations between in vivo, ex vivo and in vitro luciferase activity measurements. Ex vivo BLI demonstrated midline crossing and leakage of tumor cells. Orthotopically challenged mice followed with BLI showed an initial adaptation phase, after which imaging data correlated linearly with stereologically determined tumor dimensions. Transition from healthy to moribund state corresponded with an increasing in vivo flux but the onset of neurological deficit was clearly delayed compared to the onset of in vivo flux increase. BLI was implemented in prophylactic immunotherapy and imaging data were prognostic for therapy outcome. Three distinct response patterns were detected. Our data underscore the feasibility of in vivo BLI in an experimental immunotherapeutic setting in the GL261 glioma model.
Journal of Neuro-Oncology 10/2008; 91(2):127-39. DOI:10.1007/s11060-008-9691-5 · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.
[Show abstract][Hide abstract] ABSTRACT: The rat anterior pituitary expresses beta(2)-adrenoceptors (ARs) on somatotrophs, lactotrophs, and corticotrophs. The present study investigates whether beta(1)-ARs exist in the anterior pituitary, in which cell type(s) they are found, and whether they are regulated by glucocorticoids. As determined by quantitative RT-PCR and Western immunoblotting, the rat anterior pituitary expressed beta(1)-AR mRNA and protein. Unlike the beta(2)-AR, expression decreased to very low levels after 5-d aggregate cell culture but was strongly up-regulated in a dose- and time-dependent manner by dexamethasone (DEX). Glucocorticoids attenuated isoproterenol-induced down-regulation of beta(1)-AR mRNA levels. As examined by immunofluorescence confocal microscopy, beta(1)-AR immunoreactivity was detected in a subpopulation of gonadotrophs, but not in somatotrophs, lactotrophs, corticotrophs, thyrotrophs, or folliculo-stellate cells. beta(1)-AR-immunoreactivity cells were often surrounded by cup-shaped lactotrophs. Consistent with these findings, beta(1)-AR mRNA was considerably more abundant in the gonadotrophic alphaT3-1 and LbetaT2 cell lines than in the GHFT, GH3, and TtT/GF cell lines. DEX did not affect expression level in the cell lines. DEX also failed to up-regulate beta(1)-AR mRNA levels in aggregates from a subpopulation enriched in large gonadotrophs obtained by gradient sedimentation. In contrast, excessive DEX-dependent up-regulation of beta(1)-AR mRNA was found in a subpopulation enriched in small nonhormonal cells. The present data indicate that beta(1)-AR is expressed in a subpopulation of gonadotrophs with a topographical relationship to lactotrophs. However, the glucocorticoid-induced up-regulation does not seem to occur directly in the gonadotrophs but within (an)other unidentified cell type(s), or is transduced by that cell type on gonadotrophs.
[Show abstract][Hide abstract] ABSTRACT: Parkinson disease (PD) is a progressive neurodegenerative disorder affecting millions of people worldwide. To date, treatment strategies are mainly symptomatic and aimed at increasing dopamine levels in the degenerating nigrostriatal system. Hope rests upon the development of effective neurorestorative or neuroregenerative therapies based on gene and stem cell therapy or a combination of both. The results of experimental therapies based on transplanting exogenous dopamine-rich fetal cells or glial cell line-derived neurotrophic factor overexpression into the brain of Parkinson disease patients encourage future cell- and gene-based strategies. The endogenous neural stem cells of the adult brain provide an alternative and attractive cell source for neuroregeneration. Prior to designing endogenous stem cell therapies, the possible impact of PD on adult neuronal stem cell pools and their neurogenic potential must be investigated. We review the experimental data obtained in animal models or based on analysis of patients' brains prior to describing different treatment strategies. Strategies aimed at enhancing neuronal stem cell proliferation and/or differentiation in the striatum or the substantia nigra will have to be compared in animal models and selected prior to clinical studies.
[Show abstract][Hide abstract] ABSTRACT: Nestin is an intermediate filament protein that has originally been identified as a marker of neuroepithelial stem/progenitor cells. The present study explored whether nestin immunoreactivity (nestin-ir) is present in the rat pituitary and in which cell type(s). Nestin-ir was observed in scattered cells in the anterior, intermediate, and neural lobes. Nestin-ir cells were predominantly of stellate shape and were more numerous in immature than in adult animals. Nestin-ir did not colocalize with any pituitary hormone, and did not colocalize or only very sporadically with the folliculo-stellate cell marker S100. In the intermediate lobe, nestin-ir cells contained glial fibrillary acidic protein in an age-dependent manner. Nestin-ir cells were closely associated with endothelial and fibronectin-ir cells, but did mostly not coincide. Nestin-ir was not found in alpha-smooth muscle actin-ir myofibroblasts or in microglial cells. Regardless of age, nestin-ir was detected in some unidentifiable cells that border the pituitary cleft. Nestin-ir remained present in pituitary cultured as three-dimensional aggregates. Treatment with basic fibroblast growth factor or leukemia inhibitory factor increased the number of nestin-ir cells. Starting from anterior lobe cell monolayer cultures, nestin-ir cells could be selected and propagated to a virtually pure population. These nestin-ir cells displayed remarkable motility and proliferative activity, and did not express hormones, glial fibrillary acidic protein, or S100, but contained vimentin-, fibronectin-, and alpha-smooth muscle actin-ir. In conclusion, nestin-ir is present in the pituitary in cells that are neither hormonal nor typical folliculo-stellate. The expression pattern depends on age and lobe examined. Pericapillar localization suggests a pericyte phenotype for some of them. Whether the heterogeneous nestin-ir population also contains pituitary progenitor cells remains to be explored.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION In vivo visualization of cells by utilizing viral vector-delivered MRI reporter genes for marking cell populations (e.g. endogenous neural stem cells by expression of ferritin 1) will allow for stable cell labeling that is not lost upon cell division as compared to particle-based labeling methods. Lentiviral (LV) and adeno-associated (AAV) viral vector systems have proven very useful for stable, long term gene expression in mouse and rat brain 2,3 . Potential limitations of those systems for visualization by MRI are often insufficiently addressed. One such potential limitation is unspecific contrast by the vector itself. Here, we aim to evaluate the LV and AAV vector systems for their suitability to overexpress MRI reporter genes in mouse brain with regard to their background contrast on MRI. METHODS 3 µl of LV-eGFP, AAV-eGFP-T2A-fLuc and PBS were stereotactically injected in the striatum of C57BL/6 mice (n=5). MRI images were acquired on a 9.4T small animal scanner (Bruker Biospin) using a linear resonator (transmit) in combination with a dedicated mouse head coil (receive). T 2 -and T 2 * maps (10 TE increments, 156 x 156 μm in plane resolution, 0.8 mm slice thickness) and 3D T2*-weighted images (FLASH, TR=100 ms, TE=12 ms, flip angle 30°. 59 µm isotropic resolution) were acquired from 24 hours to up to 5 months post injection. After the last time point, animals were sacrificed, ex vivo MRI of the excised brain were acquired (also a high resolution 3D T 2 *-weighted FLASH with an isotropic resolution of 32 µm), and (immuno-) histochemical stainings were performed. RESULTS At 1day, 1 week and 1 month (shown in a) post injection, LV injection without MRI reporter genes causes clearly hypointense contrast at T2* MR at the site of injection that persists up to 5 months post injection, whereas a sham injection with PBS shows little to no contrast at the injection tract for all animals (n=10). The hypointense contrast does not correlate with transgene expression (see eGFP staining in c) but seems to correlate with the presence of Fe 3+ (as revealed by DAB-enhanced Prussian blue staining, d) and accumulation of microglial cells (CD11b staining, e). Similarly, MRI 1 week and 1 month after AAV injection result in similar MRI findings as for the LV injection (n=5).