Gene expression analysis of neuronal precursors from adult mouse brain and differential screen for neural stem cell markers

In the adult mouse brain, neuronal precursor cells continuously emanate from neural stem cells (NSC) in the subventricular zone (SVZ) and migrate into the olfactory bulb (OB) where they differentiate to serve as replenishment for GABAergic interneurons. During the migration process, PSA-NCAM (Polysialic acid-Neural cell adhesion molecule) specifically marks the neuronal precursors (PSA+ cells). This phenomenon was exploited in the framework of this doctoral thesis to isolate a homogeneous cell population of neuronal precursor cells using Fluorescence-activated cell sorting. Here, the first comprehensive picture of the gene expression in PSA+ precursors was generated using Serial Analysis of Gene Expression (SAGE). Comparison of SAGE data for PSA+ cells and for adult total brain (ATB) led to the identification of precursor-enriched genes. For selected genes, the results were validated using cDNA microarrays and quantitative real-time PCR, and the expression was analyzed at the cellular level in mouse brain using in situ hybridizations. Genes previously described in this context like the proliferation inhibitor CD24, the sialyltransferase STX and the Reelin receptor ApoER2 confirmed the identity of the precursor cells and the accuracy of the SAGE. Individual characterized genes that were so far unknown in the PSA+ cell population were identified as well as functional groups of genes by means of cluster analysis of SAGE data. The presence of transcription factors of the Sox and Dlx families, Pax6 and Meis2 indicated that secondary neurogenesis might be largely controlled by the same factors that are active during development. Clusters for apoptosis and proliferation are both upregulated. The high expression of chemotactic factors in the neuronal precursors suggests that they might be involved in neuronal cell migration. In addition, novel genes like RIKEN 3110003A17 were observed. First functional data based on the SAGE are being generated in the framework of our collaboration with the Developmental Biology Institute of Marseille. Given that a lack of markers for NSC considerably impedes progress in NSC biology, the second part of this work aimed at identifying potential NSC markers by comparing SAGE data for embryonic stem (ES) cells, PSA+ cells and ATB. The selection strategy was based on two assumptions. First, in a hierarchical order of developmental potential, ES cells are positioned above NSC, which are above restricted precursors that in turn are above adult neurons and glia. Second, the genetic programs of ES cells and NSC overlap. Thus, genes that are highly expressed in ES cells and downregulated or absent in PSA+ neuronal precursors and ATB should in part also be expressed by the few stem cells in the adult brain. Eight candidates coding for cell surface proteins were identified from the resulting list of candidates and were investigated. Due to a public database mistake in situ hybridizations were performed for the glutamate transporter GLT1 and demonstrated expression in embryoid bodies, neurospheres and, strikingly, in the SVZ, the neurogenic area of the mouse forebrain. Taken together, this doctoral thesis generated the first gene expression profile for PSA+ neuronal precursors, which -together with the SAGE library for Bruce-4 ES cells- will serve as a starting basis for future functional analysis

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

International audienceAstrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages

The Neural Circuitry of Executive Functions in Healthy Subjects and Parkinson's Disease

In our constantly changing environment, we are frequently faced with altered circumstances requiring generation and monitoring of appropriate strategies, when novel plans of action must be formulated and conducted. The abilities that we call upon to respond accurately to novel situations are referred to as ‘executive functions', and are frequently engaged to deal with conditions in which routine activation of behavior would not be sufficient for optimal performance. Here, we summarize important findings that may help us understand executive functions and their underlying neuronal correlates. We focus particularly on observations from imaging technology, such as functional magnetic resonance imaging, position emission tomography, diffusion tensor imaging, and transcranial magnetic stimulation, which in the past few years have provided the bulk of information on the neurobiological underpinnings of the executive functions. Further, emphasis will be placed on recent insights from Parkinson's disease (PD), in which the underlying dopaminergic abnormalities have provided new exciting information into basic molecular mechanisms of executive dysfunction, and which may help to disentangle the cortical/subcortical networks involved in executive processes