This idea was followed up in Chapter 5 where we aimed to silence GFAPδ in vivo in the SVZ of adult mice, a brain area with high GFAPδ expression. In contrast to the human brain, GFAPδ is expressed in all astrocytes of the developing and adult mouse brain. However, GFAPδ is an integral part of the IF network of NSCs in both species and silencing of GFAPδ is an essential tool to investigate its function in the neurogenic system in vivo. Two separate RNAi approaches, shRNA and exon skipping constructs, were designed to specifically target Gfapδ transcripts. The ability of the two RNAi approaches together with three different viral vector delivery systems was employed to silence GFAPδ expression. Both Lentivirus VSV-G and AAV5 were successfully able to transduce the SVZ. However a stable downregulation of GFAPδ failed due to an unspecific tropism of the viral particles. Moreover, off-target effects might have masked the effects of the RNAi approach in vivo. All together, this study highlights the importance of an in depth in vitroRNAi screening and proper, cell type-specific construct delivery for successful RNAi in the SVZ.
Alternatively in Chapter 6, we focused on the investigation of the regulation and function of GFAP in human NSCs. We demonstrated that Notch activity, a key regulator of NSC differentiation, controls human GFAP expression in NSCs and glial progenitors. Inhibition of Notch signaling reduced GFAP expression in undifferentiated primary human fetal NSCs (fNSCs), in vivo in zebrafish embryos, as well as in glial progenitors differentiated from immortalized fNSCs.
Differentiation of NSCs into glial progenitors was induced by human post-mortem ventricular cerebrospinal fluid, a very potent stimulus of GFAP expression in human NSCs as shown here. Upon initiation of differentiation, increased GFAP expression was associated with a decreased expression of Notch downstream targets, suggesting an inhibitory role of GFAP. Promoter assays of the Notch target gene Hes-1 confirmed a negative modulation of Notch signaling by GFAP, which is further supported by inhibition of Notch downstream target Hes-5 by GFAP overexpression. Together, this data indicates that GFAP, itself, silences Notch signaling forming a negative feedback loop which might regulate astrogenesis during brain development. Importantly, a dysregulation of GFAP expression in NSCs leads to a dramatic impairement of the sphere forming capacity of NSCs, indicating that a balanced GFAP expression is critical for NSC self-renewal.
Confirming our finding that Notch signaling is a crucial regulator of GFAP expression, we showed in Chapter 7 that an upregulation of GFAP in the context of reactive gliosis is dependent on Notch activity.
Previously our group demonstrated reduced GFAP expression upon inhibition of the proteasome. Consistantly, we report here on the ability of a proteasome activator to enhance GFAP levels. Consistent with a regulatory role of Notch, the upregulation of GFAP wasdependent on Notch activity. The proteasome is one of the major protein degradation systems in the cell and its dysregulation is implicated in many brain diseases. Intriguingly, specific inhibition of the immunoproteasome, a variant of the proteasome induced by inflammatory signaling, was sufficient to prevent Notch activity and, in turn, the upregulation of GFAP. Since recent data in our group revealed that enhanced immunoproteasome activity is characteristic for reactive astrocytes in the diseased brain, inhibition of immunoproteasome activity might represent an attractive approach to
prevent Notch activation and GFAP upregulation in reactive astrocytes. Future research will reveal whether preventing GFAP induction reduces reactive gliosis.
In the final Chapter 8, we discuss our main findings of the preceding chapters in relation to current research. We emphasize that alternative splicing of GFAP might represent an important mechanism to regulate GFAP function in different astrocyte subpopulations. Moreover, we speculate that GFAP might act as a signaling platform in the cell that binds and regulates the activity of signaling molecules in order to influence vital cellular functions.
GFAP expression is widely used as a marker for astrocytes and specific astrocyte subpopulations, such as NSCs. However, very little is known about its function and the consequences of a specialized IF network including the alternative isoform GFAPδ. Considering the widespread GFAP network as signaling platform in the cell that can regulate signaling pathway activities, will significantly enhance our knowledge on the function of the broadly used astrocyte marker GFAP.