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6.4.1 Ablation of Brg1 protein in adult neural stem cells

In order to address the function of Brg1 in adult neural stem cells, the Brg1 protein was ablated by using an inducible GLASTCreERT2_{line to specically delete the pro-} tein in GLAST expressing astrocytes that are described to contain the multipotent stem cells in the adult neurogenic regions [Ninkovic and Gotz, 2007; Jackson and Alvarez-Buylla, 2008]. Supported by previous studies it became clear that Brg1 holds an important function in embryonic neural stem cells as reported by Matsumoto et al. (2006). The researchers claimed that stem cell maintenance is mediated by Brg1 and later during dierentiation it seemed to be important for glial dierentiation rather than neuronal dierentiation. The two temporal as well as spatial indepen- dent roles of Brg1 were also well described by Lessard et al. (2007). They reported that a switch in the subunit composition of Brg1-containing complexes may pro- mote neural stem cell self-renewal/proliferation. The specialized function of Brg1 in neural stem cells was likely mediated through its interaction with the progenitor- specic subunits of the SWI/SNF chromatin remodeling complexes. The transition from proliferative to neurogenic divisions required the exchange of these subunits specic for stem cell maintenance and thereby promoting neuronal dierentiation. Therefore two dierent, very specic Brg1 containing complexes appeared at two distinguishable time points in embryonic neurogenesis [Lessard et al., 2007] and it

is believed that Brg1 is required for neuronal dierentiation [Seo et al., 2005b]. An antagonist of Brg1 function with regard to neuronal dierentiation was shown to be Geminin, a novel coiled-coil protein which was previously characterized to play a role in maintenance of genome integrity through regulation of DNA replication licensing [Seo and Kroll, 2006]. Geminin was found to be highly expressed in embry- onic neuronal progenitor cells where it blocks the association of Brg1 with proneural genes, such as Neurogenin or NeuroD [Seo et al., 2005b]. In dierentiating neurons Gem levels diminish and allow Brg1 to promote neuronal dierentiation [Seo et al., 2005a]. However, the function of Brg1 containing complexes and the interaction with other regulatory proteins in adult neurogenesis was so far unknown.

By analyzing Brg1 oxed mice crossed with GLASTCreERT2 _{and a reporter line} (Z/EG) to follow the recombined cells after tamoxifen induction in the adult brain at 3 dierent survival times an important phenotype in neuronal dierentiation could be detected. The Brg1 protein was not completely disappeared 6 days after tamoxifen induction (Figure 5.44), but after 14 days Brg1 protein was no longer detectable in GFP+ recombined cells (Figure 5.46). GFP+ Dcx+ cells were slightly reduced at the SEZ in Brg1 oxed mice suggesting a reduction in the number of newly generated neuroblasts 14 days after tamoxifen induction. GFP+ cells entering the RMS to migrate to the OB seemed to disperse and migrate into other areas, such as the nearby white matter or the striatum (Figure 5.47 b). The few recombined cells that managed to reach the OB were often not neuronal as they did not con- tain Dcx but started to express glial markers such as NG2 (Figure 5.48 a,b) and also showed a clear change in cell morphology as the cells positive for GFP had a branched process formation characteristic for oligodendrocyte progenitors (Figure 5.47 c-g). The quantication of GFP+Dcx+ cells in the OB reected a signicant reduction in Brg1 oxed mice from ≈ 85 % to 40% suggesting the possibility that

Brg1 is important for neuronal dierentiation in the adult SEZ/RMS/OB system (Figure 5.48 c,d). The DG remains to be examined in the future to compare the observed eects after Brg1 deletion in both neurogenic areas.

The third time point analyzed 28 days after tamoxifen application conrmed the observations made already after 14 days. Moreover, the observed change in Brg1 oxed mice became more prominent as very few cells reached the OB and none of the GFP+ cells migrated toward the GL of the OB to become integrated in the neuronal circuitry (Figure 5.49). Furthermore, the expression of glial markers such as NG2 was also more prominent in the longer survival analysis than after 14 days

(Figure 5.50 a,b).

In order to study adult neural stem cells derived from the SEZ of Brg1 oxed mice were infected with a Cre-containing retrovirus to delete Brg1 in all dividing cells in the oating state. The control cultures were not infected at all. The number and size of neurospheres were reduced in cultures where Cre led to the ablation of Brg1 protein (Figure 5.52). This observation was completely consistent with the ndings that Lessard et al. (2007) reported. Their in vitro analysis was done with embryonic neurospheres from WT and Brg1 oxed mice crossed with a nestinCre mouse line to specically delete Brg1 protein in cells expressing nestin starting at E10.5. When they analyzed the number and size of the neurospheres, both were signicantly reduced in Brg1-decient cultures compared to WT cultures [Lessard et al., 2007] after 7 days, suggesting a crucial role for Brg1 in stem cell maintenance and a potent function in embryonic neurogenic precursors as well as in adult neu- rogenic precursors. As Brg1 was found to be expressed in almost all cells in adult neural stem cell cultures it may also play a role in neuronal dierentiation in vitro as it was shown in vivo. Cells were infected with a Cre-containing retrovirus 2 hours after plating. After 7 days of dierentiation there was a signicant change from neuronal to more oligodendrocytic dierentiation (Figure 5.53). Taken together, Brg1 seems to be important for neural stem cell maintenance and neuronal dier- entiation both in vitro and in vivo. When Brg1 was deleted cells were not able to proliferate in a normal frequence. During neuronal dierentiation a switch from the neurogenic to the gliogenic pathway occurred. Besides the importance of Brg1 containing complexes with specic subunit presence in either neural stem cells or more dierentiated neurons [Lessard et al., 2007], so far little is known about the function of Brg1 in a complex mechanism such as adult neurogenesis. This study shed some light on the importance of Brg1 in this regard but further examinations will be carried out to address the functions more precisely.