Prof. Dr. Andreas Faissner, Cellmorphology and Molecular Neurobiology, Faculty of Biology and Biotechnology
Research Programme:
Molecular and cellular bases of development, plasticity and regeneration of neural tissues
The central nervous system presents a daunting complexity and variety of cell types and axonal connections. The laboratory aims at the elucidation of the molecular and cellular bases of the regulatory processes involved in the generation of this system. The directing concept posits that an understanding of developmental processes will shed light on phenomena such as the plasticity of CNS structures, basic mechanisms of neural degeneration, and novel perspectives on cellular repair in the broader framework of regenerative medicine.
The laboratory devotes a special interest to the molecules involved in cellular interaction, namely cell adhesion molecules (CAMs) and the extracellular matrix (ECM). These molecules are investigated using techniques of mouse genetics (transgenic mice and knock-out animals), molecular and cell biology, immunology with production of monoclonal antibodies, biochemistry and imaging including fluorescence, confocal laser scanning and digital video microscopy.
The Department of Cellmorphology and Molecular Neurobiology is divided in several thematic groups that focus i) on the biology of neural stem cells, radial glia stem cells, motoneuron and glial progenitors and their microenvironment, the stem cell niche during development and under pathological conditions; ii) on neuron-glia interactions and their regulatory influence on axon growth and guidance, as well as synaptogenesis and synaptic plasticity; iii) on the generation of retinal stem cells, their progeny, the generation of retinal progenitors and subpopulations, their integration into neural networks and the possible application to visual system lesions and degeneration.
The laboratory has initiated a systematic characterisation of the neural stem cell niche. ECM molecules constitute an essential building block and intervene in maintenance and differentiation of neural stem cells (NSCs). Further on, data have been collected that support regulatory roles of receptor protein tyrosine phosphatases (RPTPs) in retinogenesis and stem cell regulation. With regard to the response of neural cell types to ECM constituents, a special emphasis is given to small GTPases, key regulators of intracellular processes. Models for ECM molecules include the tenascin gene family and their transcriptional regulators, e.g. Pax6; the condroitinsulfate proteoglycan Phosphacan, complex carbohydrates such as the DSD-1- and the MAb 4860 and MAb 5750 carbohydrates, and RPTP-b/z and related genes.
Important objectives are the identification of ECM receptor systems of functionally interesting domains and the elucidation of signal transduction pathways downstream. Theses studies eventually will enhance our knowledge about the molecular architecture of the neural microenvironment and help to answer the questions concerning its instructive potential.
The laboratory offers training opportunities in the three thematic groups. Currently, the work is supported by grants from the German Research Council (DFG), the Federal Ministry of Education, Research and Technology (BMBF), and by the Land NRW.
Outline of the research project
Molecular Structure and functions of the neural microenvironment
The pericellular environment makes up for 25% of the developing brain volume. The environment is pervaded with a structured extracellular matrix (ECM). This biochemical compartment consists of an exquisite network of glycoproteins and proteoglycans. Astrocytes represent the most important source of ECM components in the CNS. This cell type plays a pivotal role throughout development and in the adult CNS. Thus, current views suggest that radial glia in the developing and astrocytes in the adult CNS act as neural stem cells. Once formed, neurons rely on astroglial cells in the context of radial glia guided migration, and axon growth and guidance. Astrocytes also play a central role in the generation and plasticity of functional synapses. Finally, astrocytes form glial scars upon lesion that seal CNS territories, but likewise also generate obstacles to axonal regeneration.
Glycoproteins and proteoglycans of the extracellular matrix (ECM)
The laboratory has contributed to the discovery of central constituents of the ECM of the neural stem cell niche during development and in the realm of adult neuronogenesis. The models the laboratory studies in more detail are tenascin-C (Tnc) and the chondroitinsulfate proteoglycan (CSPG) phosphacan. Tenascin-C glycoproteins represent one of the most abundant glycoproteins of the neural ECM. TN-C glycoproteins are transiently expressed by astrocytes during CNS development, consist structurally of EGF-type repeats, fibronectin-type-III (FNIII) modules and homologies to fibrinogen. Many isoforms are generated in the context of a binary combinatorial code of alternatively spliced FNIII-domains. Tnc is expressed in the neural stem cell niche and influences the maturation of neural stem cells (NSCs) and various progenitors. The CSPG phosphacan represents a splice variant of receptor phosphotyrosine phosphatase (RPTP) beta/zeta. This receptor is expressed by glial cells and occurs in two isforms named RPTP-beta(l) and RTP-beta(s). Phosphacan binds to TN-C and the RPTP-beta isoforms might function as glial transmembrane receptors of the glycoprotein. The CSPG carries the DSD-1-epitope, a glycosaminoglycan modification that is specifically recognized by mAb 473HD and is by itself sufficient to stimulate neurite outgrowth. Recently, we could show that the DSD-1-epitope is a novel surface marker that can be used to purify NSCs by immunological methods. Tnc and RPTP are also expressed at later stages of development, where they may play a role in axon growth and guidance, and neural cell migration. Although largely absent from adult CNS, both components are upregulated in various lesioning models, which suggest a close association with the reorganisation of tissues.
Receptor systems and signalling mechanisms
The structure-function analysis of Tnc and RPTP is carried out using monoclonal antibodies, recombinantly expressed domains and in vitro assay systems for NSC differentiation, axon growth and guidance, and synaptogenesis. In vivo, the analysis is complemented by the study of transgenic models of ECM expression and function. Important objectives are the identification of receptor systems of functionally interesting domains, and the elucidation of signal transduction pathways downstream. In this context, the laboratory has embarked in the systematic analysis of tyrosine phosphatases and their influence on neural differentiation. Theses studies eventually will enhance our knowledge about the molecular architecture of the neural microenvironment and help to answer the questions concerning its instructive potential.
Molecular architecture of the neural stem cell niche
One line of research concerns the issues of ECM-dependent regulation of gene expression in the neural stem cell niche. This project involves the analysis of gene-trap stem cell lines of the nervous system. A second topic exploits the immunoisolation protocol for NSCs that has been developed by the laboratory. Resulting populations will be investigated by transplantation paradigms in adequate models including laser lesions of the cortex (in collaboration with Prof. Eysel and Dr. Mittmann, Ruhr-University). Finally, the laboratory is committed to the analysis of the roles GTP-binding proteins and their regulators play in stem cell maturation and differentiation.
Techniques
- Primary culture of defined neural cell types
- In vitro bioassays for determination of cell adhesion, cell repulsion and neurite outgrowth
- Video-microscopy of life primary neural cell cultures
- Generation of monoclonal antibodies to neural ECM molecules
- Purification and biochemical characterisation of neural ECM componentsand complementary receptors
- Molecular cloning of neural ECM components, expression of fusionproteins, in situ hybridisation
- Functional characterisation of ECM constituents in transgenic andknock-out animals
- Morphological analysis using light, immunofluorescence, electron,and confocal laser scanning microscopy
Courses
- Andrea Horvat-Bröcker and Andreas Faissner - "Analysis of tyrosine phosphatases by RT-PCR and in situ hybridization"
- Swetlana Sirko and Andreas Faissner - "In vitro culture and analysis of neurospheres"
- Martin Pyka, Melanie Michele and Andreas Faissner - "In vitro culture and morphometric analyis of primary rat embryonic hippocampal neurons"
- Sören Moritz and Andreas Faissner - "Digital videomicroscopy of life cells in vitro"
- Alice Klausmeyer, Witold Mütze and Andreas Faissne - "Analysis of biological specimen by laser scaning microscopy"
- Witold Mütze, Nicole Brösicke and Andreas Faissner - "In vitro models of glioma cell culture and migration"
- Witold Mütze, Marion Voelzkow and Andreas Faissner - "Basics of electron microscopy"