DNA topoisomerase II\u03b2 stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression

Merve Zaim,Sevim Isik

doi:10.1186/s13287-018-0859-4

Abstract

BackgroundDNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases.Methods and resultsFor this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ.ConclusionOur results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Highlights

DNA topoisomerase IIβ is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance
Results Modified N3 medium (mN3) cytokine combination induced successful neural differentiation in Human mesenchymal stem cell (hMSC) To evaluate the neural differentiation of the bone marrow-derived hMSC (BM-hMSC) line, we initially demonstrated that our data were in agreement with the criteria of the International Society for Cellular Therapy
For characterization of the hMSC line, the immunophenotypic cell surface profile for CD45, HLA-DR, CD105, CD34, CD73, and CD90 were analyzed by flow cytometry; cells were positive for the markers CD73, CD90, and CD105, and negative for CD45, CD34, and HLA-DR (Additional file 1: Figure S1)

Summary

Introduction

DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. Topo IIβ may be involved in the regulation of certain gene expressions required for neurite outgrowth during neural differentiation [10, 12, 13]

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