Interactions between mouse CNS cells : microglia and neural precursor cells

Abstract

The mammalian central nervous system (CNS) contains a variety of cells, all specialized to perform different functions. Most numerous are the three types of glia cells, whose basic role is to support the signaling units of the CNS, the neurons.

The perspective of this thesis is from the glia cells; particularly the microglia cells, a non-neural population of cells that are spread throughout the CNS. In the healthy CNS these cells are resting, but as a consequence of various CNS disturbances they rapidly become activated, frequently together with astrocytes, the second type of glia cells (oligodendrocytes being the third). This can happen slowly, as in neurodegenerative diseases, or quickly as in acute CNS lesions such as stroke. Both processes involve microglia cells, and sometimes macrophages from the circulation. These latter cells are difficult to distinguish from the microglia cells.

In an effort to generate microglia specific markers we used phage display technology to select microglia specific peptides from a random peptide library displayed on the phage surface. Two sets of selection strategies were compared with regard to phage-clone enrichment. The first strategy was based on phage binding to monolayers of primary microglia fixed to a solid surface, while the second strategy was based on fluorescence activated cell sorting (FACS) of microglia cells with bound phages. The latter protocol was found to be superior. Five phage-clones that preferentially bound to microglia cells were isolated. One of the selected clones was shown to be microglia specific by free peptide inhibition and selective in binding to microglia cells, as compared to blood-derived monocytes.

Much of our current knowledge in neurobiology derives from studies of cells in culture, a less complex substitute for in vivo studies. As a bridge between monolayer CNS cell cultures and in vivo animal models we set up a three-dimensional culture system, so called aggregate cultures from mouse CNS cells. These aggregates were characterized in detail regarding cellular composition and dynamics, as well as the expression of several neuropeptides and neurotransmitters. All the principle brain cells were present in the aggregates and their numbers changed over time, neurons being the most numerous. The cells appeared to mature as judged by their morphology and, in the case of neurons, the increased expression of synapse specific proteins. Among the investigated neuropeptides, enkephalin and dynorphin were the most abundant followed by galanin, approximating their expression in CNS development. We also found that neural precursor cells, capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, were maintained in the aggregates, even after more than two months of culturing. Treating the aggregates with EGF led to the formation of an outer layer of nestin-positive precursor cells.

Using the aggregate culture in part, we found that factor(s) secreted from microglia cells attracts neural precursor cells in a chemotactic manner. This finding may explain the preferred migration of precursor cells to sites of CNS injury. Furthermore, microglia derived factors could affect the differentiation of neural precursor cells, such that more neurons were formed. Together these results suggest important functions of microglia cells in CNS development and pathology. It is reasonable to believe that the migration of neural precursor cells is directed both by attractant and repellant cues. Reactive astrocytes are well known to inhibit growing axons and recently also suggested to inhibit the migration of neural precursor cells. We show that astrocytes in culture repel neural precursor cells and that this effect is mediated by secreted Slit proteins. This conclusion is based on several observations; astrocytes produce Slit and the astrocyte-repellant effect was blocked by the ectodomain of the Slit receptor, and finally, recombinant Slit could substitute for astrocyte derived Slit. Knowledge about the interplay between attractive and repulsive cues may be important for the manipulation of neural precursor cells for medical purposes.