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Abstract

During the development of the central nervous sytem (CNS) the maturation of oligodendrocytes occurs through the tightly regulated activity of diverse intrinsic and extrinsic signalling factors. Our group identified the STAR-family protein Sam68 as one of those intrinsic cues involved in oligodendrocyte differentiation. The level of Sam68 increases with ongoing maturation and it regulates the expression of Myelin Basic Protein (MBP). Despite its role in OPC (oligodendrocyte precursor cells) maturation, previous studies already identified Sam68 as a promotor of neural stem cell (NSC) differentiation and as a Tenascin-C- regulated target gene. However, the mechanism(s) how Sam68 regulates NSC and particularly oligodendrocyte development remained incompletely understood. This thesis provides completely new insights into the role of Sam68 during forebrain and particularly, oligodendrocyte development. Furthermore, my results exhibit hnRNPA1 as a new interaction partner of Sam68 in oligodendrocyte development and provide a basic concept for the regulation of MBP-expression through both proteins. Our group already investigated the general expression pattern of the three STAR-family members, Sam68, Slm-1 and Slm-2 and showed a specific expression of all three proteins during forebrain development. The present thesis characterised for the first time the identity of Sam68 expressing cells during forebrain development. Neuroepithelial, radial glia cells and their derivative cell types were exhibited to express Sam68. Furthermore, time-dependent cell culture experiments revealed a significant shift in the expression pattern towards differentiating cells. These findings supported earlier studies of our group indicating a promotive role of Sam68 in cell differentiation. The second part describes a successfully established method for the high efficiency transfection of non-adherent primary rat OPCs. This new transfection protocol enables for the first time the reproducible transfection of non-adherent OPCs with a high viability, a regular maturation pattern and an acceptable transfection efficiency. Regarding the time-consuming and low yield isolation of OPCs, this method represents a big advantage and provides the basis for the main goal of this thesis, which displays the determination of the role of Sam68 during oligodendrocyte development. Although oligodendrocytes count to the best characterised cell types within the CNS, many intracellular signalling pathways regulating their development and differentiation remained elusive. Here, I discovered the respective Sam68 domains which modulates cell growth, MBP-expression and myelin-sheet formation. The complete RNA-binding domain and the NLS-sequence were shown to be relevant for the regulation of MBP-expression as well as for the formation of myelin-sheets. The relevance of the NLS-sequence and the knowledge about Sam68 as a regulator of alternative splicing events led to the assumption that Sam68 may regulate MBP-expression through modulating its splicing. The well-studied splicing regulator hnRNPA1 was already shown to regulate the alternative splicing of myelin-associated glycoprotein in cooperation with the STAR-family member Quaking I. Thus, I assumed a similar interaction of Sam68 and hnRNPA1 in the regulation of MBP expression. Indeed, MBP-level was downregulated after an hnRNPA1 knockdown and this effect was intensified after an additional overexpression of Sam68. These results provide a good basis to unravel the very complex regulation between Sam68 and hnRNPA1 in oligodendrocyte development.