Localization of Endogenous Biotin-Containing Proteins in Mouse Bergmann Glial Cells

A peroxidase-conjugated avidin–biotin complex was used to detect endogenous biotin-containing proteins in mouse cerebellum. By this method, Bergmann glial cells were found to be strongly labelled in the adult mouse cerebellum. Developmentally, cells in the granular layer, probably astrocytes, appeared to be labelled around postnatal 10-day (P10). Their labelling decreased after P20, although the positive-labelling remained in the Bergmann glial cells up to the adult stage. The findings were confirmed by using a Alexa Fluor 488-conjugated streptavidin technique. The labelling was not affected by routine hydrogen peroxide treatment, but it was eliminated by avidin–biotin blocking. By another transblot method, the reactive proteins in the mouse cerebellum were found to be 120?kDa (the strongest one) and 75?kDa. For electron microscopy, a gold-conjugated anti-biotin antibody was immunoreacted to the mitochondria of Bergmann glial cells. These results suggest that endogenous biotin-containing proteins are abundant in the Bergmann glial cells. Therefore, the avidin–biotin complex method is useful for detecting Bergmann glial cells, probably because of the difference of biotin metabolism in the cerebellar glial cells.     

Single-Cell Transcriptional Profiling Reveals Cellular Diversity and Intercommunication in the Mouse Heart

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GLAST Activity is Modified by Acute Manganese Exposure in Bergmann Glial Cells

Glutamate is the major excitatory amino acid neurotransmitter in the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed in neurons and glial cells. Overactivation of glutamate receptors results in neuronal death, known as excitotoxicity. A family of sodium-dependent glutamate transporters enriched in glial cells are responsible of the vast majority of the removal of this amino acid form the synaptic cleft. Therefore, a precise and exquisite regulation of these proteins is required not only for a proper glutamatergic transmission but also for the prevention of an excitotoxic insult. Manganese is a trace element essential as a cofactor for several enzymatic systems, although in high concentrations is involved in the disruption of brain glutamate homeostasis. The molecular mechanisms associated to manganese neurotoxicity have been focused on mitochondrial function, although energy depletion severely compromises the glutamate uptake process. In this context, in this contribution we analyze the effect of manganese exposure in glial glutamate transporters function. To this end, we used the well-established model of chick cerebellar Bergmann glia cultures. A time and dose dependent modulation of [³H]-d-aspartate uptake was found. An increase in the transporter catalytic efficiency, most probably linked to a discrete increase in the affinity of the transporter was detected upon manganese exposure. Interestingly, glucose uptake was reduced by this metal. These results favor the notion of a direct effect of manganese on glial cells, this in turn alters their coupling with neurons and might lead to changes in glutamatergic transmission.

     

FGF/FGFR2 Signaling Regulates the Generation and Correct Positioning of Bergmann Glia Cells in the Developing Mouse Cerebellum

The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse.     

Transcriptional Differences between Normal and Glioma-Derived Glial Progenitor Cells Identify a Core Set of Dysregulated Genes

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Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Co-expression Profiling of Autism Genes in the Mouse Brain

Autism spectrum disorder (ASD) is one of the most prevalent and highly heritable neurodevelopmental disorders in humans. There is significant evidence that the onset and severity of ASD is governed in part by complex genetic mechanisms affecting the normal development of the brain. To date, a number of genes have been associated with ASD. However, the temporal and spatial co-expression of these genes in the brain remain unclear. To address this issue, we examined the co-expression network of 26 autism genes from AutDB (http://mindspec.org/autdb.html), in the framework of 3,041 genes whose expression energies have the highest correlation between the coronal and sagittal images from the Allen Mouse Brain Atlas database (http://mouse.brain-map.org). These data were derived from in situ hybridization experiments conducted on male, 56-day old C57BL/6J mice co-registered to the Allen Reference Atlas, and were used to generate a normalized co-expression matrix indicating the cosine similarity between expression vectors of genes in this database. The network formed by the autism-associated genes showed a higher degree of co-expression connectivity than seen for the other genes in this dataset (Kolmogorov–Smirnov P = 5×10⁻²⁸). Using Monte Carlo simulations, we identified two cliques of co-expressed genes that were significantly enriched with autism genes (A Bonferroni corrected P<0.05). Genes in both these cliques were significantly over-expressed in the cerebellar cortex (P = 1×10⁻⁵) suggesting possible implication of this brain region in autism. In conclusion, our study provides a detailed profiling of co-expression patterns of autism genes in the mouse brain, and suggests specific brain regions and new candidate genes that could be involved in autism etiology.     

Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy.