Mammalian Numb-interacting Protein 1/Dual Oxidase Maturation Factor 1 Directs Neuronal Fate in Stem Cells

Abstract

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helixloop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Reactive oxygen species and the neuronal fate

“…Nip and Duox are expressed in the neuronal lineage in stem cells as well as the developing brain. Depletion of nip1 expression by short hairpin RNA led to reductions in neuronal differentiation and ROS generation [53]. Since H 2 O 2 is known to stimulate neuronal differentiation [71][72][73][74][75], our work provided the first demonstration that Nip can regulate neurogenesis in stem cells by regulating the cellular levels of H 2 O 2 .…”

“…What remains puzzling are the sheer number of enzymes (different Nox and Duox isoforms) expressed in neural progenitor cells, all of which are capable of producing ROS. Studies in mouse tissues and mouse embryonic stem cells also reveal an abundance of these enzymes in these systems [49,50,53]. Attempts at characterizing Nox and Duox proteins suggest differences in temporal expression during differentiation, as well as differences in the resulting phenotypes from their knockdown or overexpression.…”

“…Duox1 and Duox2 (and their respective maturation factors) were found to be expressed in a variety of adult tissues including the thyroid, airway tracts, salivary glands and the gastro-intestinal tract [52]. Recently, our laboratory detected these enzymes in the developing embryonic and postnatal mouse brain [53]. The structure of Doux features a canonical C-terminal Nox catalytic domain, a bis-heme peroxidase-like domain, and a Ca 2?…”

“…Since H 2 O 2 is known to stimulate neuronal differentiation [71][72][73][74][75], our work provided the first demonstration that Nip can regulate neurogenesis in stem cells by regulating the cellular levels of H 2 O 2 . Hence, we proposed a new mechanism that guides neuronal differentiation and characterized Nip1-mediated ROS production as a novel intrinsic regulator of neuronal cell fate in stem cells [53].…”

Redox-regulated fate of neural stem progenitor cells

“…DUOX maturation factor, DUOXa1 (also known as a Numb-interacting protein 1) was described as an intrinsic regulator of neuronal fate in stem cell differentiation [107]. DUOXa1 is highly expressed in undifferentiated NSPCs and plays a role in DUOX1-dependent superoxide and H 2 O 2 generation.…”