Stat1 Regulates Neural Stem-Cell Function By Transcriptional Regulation of Sox9

Background: Neural stem cells (NSCs) have great potential in treating neurodegeneration. The self-renewal of endogenous NSCs can be increased during acute injury. We have shown that during EAE (the animal model of multiple sclerosis) chronic inflammation decreases the repair potential of subventricular zone (SVZ) NSCs. However, the specific pathways that are altered are largely unknown.

Objectives: To identify mechanisms for neural stem cell failure in EAE.

Methods: We investigated the expression of STAT1 by qPCR and confocal microscope in EAE. Retrovirus STAT1-GFP to study self-renewal of NSCs. Stat1-/- NSCs were analyzed in vivo and in vitro. Microarray analysis of IFN-g treated and Stat1-/- NCSs with genomic pathway analysis,  was used to identify new candidates and confirmed by western blot. Chromatin Immunoprecipitation (CHIP) assay and transfection of Sox9 luciferase constructs were used to refine regions of Sox9 involved in Stat1 binding. 

Results:  We report increase of STAT1 in the Sox2+ adult NSCs from SVZ of mice in the chronic and relapsing remitting models of EAE. Overexpressing STAT1 in NSCs by a Stat1 virus decreases NSCs self-renewal capacity; in contrast Stat1 -/- mice exhibit an increase in proliferation and population doublings in neurospheres in vitro and an increase in the proliferation of Sox2 NSCs in vivo.  Furthermore, Stat1-/- NSCs showed increased self-renewal, neurogenesis, oligodendrogenesis and survival to deleterious signals. Activation of STAT1 during EAE is likely mediated by IFN-g as evidenced by increased IFN-g and not IL-17 producing cells in the CSF of EAE animals. In vitro, IFN-g was superior to IL-17 in reducing self-renewal of NSCs, inducing STAT1 phosphorylation and increased Stat1, p21Cip1, p16ink4A gene expression. Using genomic network analysis of IFN-g treated NSCs, we found that Stat1 gene network was induced by IFN-g in NSCs, and Stat1^-/- NSCs were resistant to the IFN-g deleterious effect. One of the increased genes in Stat1-/- NSCs was the core NSC transcription factor Sox9, critical for NSCs renewal and differentiation. IFN-g decreases Sox9 expression in a Stat1 dependent manner. Furthermore Sox9 upregulation associates with increased self-renewal and down-regulation with decreased self-renewal of NSCs.  CHIP assay, revealed that Stat1 binds three different regions of the Sox9 promoter. Using multiple Sox9 luciferase constructs spanning the entire promoter region to refine the Sox9 region bound by Stat1, we demonstrated that Stat1 is a transcriptional repressor of Sox9 with maximum efficacy in a conserved 0.5 Kb region of the 5’ flanking region of the Sox9 promoter.

Conclusions:

We identify for the first time an immunological mechanism that negatively impacts NSCs, and  a novel role of Stat1 as a checkpoint for self-renewal of NSCs limiting homeostatic proliferation. Thus, inactivation of Stat1 and promotion of Sox9 may offer a target to enhance neuro-regeneration.