Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate.

Neural stem cell (NSC) maintenance and functions are regulated by reactive oxygen species (ROS). However, the mechanisms by which ROS control NSC behavior remain unclear. Here we report that ROS-dependent Igfbp2 signaling controls DNA repair pathways which balance NSC self-renewal and lineage commitment. Ncf1 or Igfbp2 deficiency constrains NSCs to a self-renewing state and prevents neurosphere formation. Ncf1-dependent oxidation of Igfbp2 promotes neurogenesis by NSCs in vitro and in vivo while repressing Brca1 DNA damage response genes and inducing DNA double-strand breaks (DDSBs). By contrast, Ncf1-/- and Igfbp2-/- NSCs favor the formation of oligodendrocytes in vitro and in vivo. Notably, transient repression of Brca1 DNA repair pathway genes induces DDSBs and is sufficient to rescue the ability of Ncf1-/- and Igfbp2-/- NSCs to lineage-commit to form neurospheres and neurons. NSC lineage commitment is dependent on the oxidizable cysteine-43 residue of Igfbp2. Our study highlights the role of DNA damage/repair in orchestrating NSC fate decisions downstream of redox-regulated Igfbp2.

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b.

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.

Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity.

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.