The Redox Function of APE1 Is Involved in the Differentiation Process of Stem Cells toward a Neuronal Cell Fate

Carlo Vascotto,Giuseppe Gianfranceschi,Carlo Alberto Beltrami,Milena Romanello,Massimo Faggiani,Antonio Paolo Beltrami,Mark R. Kelley,Daniela Cesselli,Piercamillo Parodi,Rossana Domenis,Gianluca Tell,Giovanna Vagnarelli,Silvia Rigo,Natascha Bergamin

doi:10.1371/journal.pone.0089232

Carlo Vascotto, Giuseppe Gianfranceschi + Show 12 more

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https://doi.org/10.1371/journal.pone.0089232

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Abstract

Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. Methods and results: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

Highlights

APE1/Ref-1 (Apurinic apyrimidinic Endonuclease/Redox effector factor 1, called APEX1 or Ref-1 and here referred to as APE1) the mammalian ortholog of E. coli Xth (Exo III), is a master regulator of cellular response to oxidative stress and plays a central role in the maintenance of genome stability and transcriptional regulation
NTERA-2 cl.D1 (NT2-D1) cells significantly increased their positivity to Microtubule Associate Protein2 (MAP2) and glial fibrillary acidic protein (GFAP), known markers of neural and glial cells, respectively
This work has several aspects of novelty that have been summarized in Figure 7D: i) APE1 accumulates, in a ROSdependent fashion, into the nuclei and associates to the chromatin of adult stem cells differentiated towards a neuronal fate, ii) the redox function of APE1 plays a role in the neuronal differentiation of both adult and embryonic stem cells, and iii) the use of a specific inhibitor of the redox function of APE1 increases the extent of neuronal differentiation, facilitating the differentiation towards specific neuronal subsets

Summary

Introduction

APE1/Ref-1 (Apurinic apyrimidinic Endonuclease/Redox effector factor 1, called APEX1 or Ref-1 and here referred to as APE1) the mammalian ortholog of E. coli Xth (Exo III), is a master regulator of cellular response to oxidative stress and plays a central role in the maintenance of genome stability and transcriptional regulation. APE1 has another major cellular function, since it works as a reduction-oxidation (redox) factor and stimulates the DNA binding activity of several transcription factors that are involved in cell proliferation and differentiation. This function is accounted for by the redox sensitive Cys. We recently provided evidence that this redox regulation of APE1 may impact on protein subcellular mitochondrial trafficking [10] In this regard, the specific block of APE1 redox activity on NF-kB with E3330 impairs hemangioblast development in vitro [11], confirming the leading role of APE1 redox function in affecting the cell differentiation programs. It has been demonstrated that APE1 acts as a cleansing factor of abasic rRNA and is able to bind hairpin structures of RNA molecules [16,17]

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