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Abstract

Chondroitin sulfate proteoglycans (CSPGs) and their specific sulfation pattern by chondroitinsulfotransferases (Chsts) appear to play a crucial role for the behaviour of neural stem cells (NSCs) in the embryonic neural stem cell niche during mouse forebrain development. It has been shown that the inhibition of the sulfation by sodium chlorate or the degradation of the CSPG glycosaminoglycans (GAGs) by chondroitinase ABC leads to less proliferation and altered cell fate decisions of the NSCs (Sirko et al., 2007, Akita et al., 2008, Sirko et al., 2010).

In the present study, the impact of the overexpression of one specific Chst on the behaviour of NSCs was examined. Therefore, Ust was overexpressed in in vitro and in vivo experiments in order to elucidate the role of disulfated CS-units on NSCs' self-renewal, proliferation as well as differentiation.

The functionality of the Ust fusion proteins (Ust, Ust mut) and their correct location to the Golgi apparatus, where the sulfation of the GAGs takes place, could be confirmed by CS-GAG epitope detection with the monoclonal antibody 473HD and with the Golgi apparatus marker transgolgin97.

Initially, the effect of Ust overexpression on the differentiation and proliferation behaviour of neurosphere-derived NSCs was examined by the respective in vitro assays. Here, I could observe an increase in neurogenesis. The enhanced neuronal differentiation occurred at the expense of oligodendrocyte precursor cell (OPC) generation, while NSC proliferation and self-renewal were not effected. Although, there was a very small but significant increase in the number of neurospheres after Ust overexpression without growth factor supplementation.

To verify, whether Ust overexpression stimulates neurogenesis similarly in vivo, I performed in utero electroporation experiments. The immunohistochemical analysis of in utero electroporated E14.5 mouse cortices two days post-transfection revealed that Ust overexpression caused a thinner cortical plate, which is a consequence of reduced numbers of NeuN+-neurons in this area. In contrast, the amount of Pax6+ radial glia cells in the ventricular zone was slightly increased. The analysis of NSC proliferation in vivo by EdU incorporation did not reveal a significant difference and was consistent with the obtained in vitro results.

Moreover, I designed a strategy for an Ust conditional knockout mouse line to enable future examinations of the sulfation pattern impact on CNS development.

In conclusion, I could show an efficient and functional Ust overexpression, which exhibited an effect on NSC behaviour in vitro and in vivo. The effect is due to the modified sulfation pattern, which could be confirmed by using the negative control Ust mut. Consistent with previous observations, the sulfation of the CSPGs plays a role in the commitment of NSCs within the NSC niche and could function as a possible communication platform between NSCs and their extracellular surrounding in the NSC niche.