Lingo-1 shRNA and Notch signaling inhibitor DAPT promote differentiation of neural stem/progenitor cells into neurons

doi:10.1016/j.brainres.2015.11.029

Brain Research

Volume 1634, 1 March 2016, Pages 34-44

https://doi.org/10.1016/j.brainres.2015.11.029 Get rights and content

Highlights

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Silencing Lingo-1 shRNA could promote the cells differentiate more into neurons.
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Blocking Notch signaling could promote the cells differentiate more into neurons.
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The stem/progenitor cells expressed more Lingo-1 after treated with DAPT.

Abstract

Determination of the exogenous factors that regulate differentiation of neural stem/progenitor cells into neurons, oligodendrocytes and astrocytes is an important step in the clinical therapy of spinal cord injury (SCI). The Notch pathway inhibits the differentiation of neural stem/progenitor cells and Lingo-1 is a strong negative regulator for myelination and axon growth. While Lingo-1 shRNA and N-[N-(3, 5-difluorophenacetyl)-1-alanyl]-S-Phenylglycinet-butylester (DAPT), a Notch pathway inhibitor, have been used separately to help repair SCI, the results have been unsatisfactory. Here we investigated and elucidated the preliminary mechanism for the effect of Lingo-1 shRNA and DAPT on neural stem/progenitor cells differentiation. We found that neural stem/progenitor cells from E14 rat embryos expressed Nestin, Sox-2 and Lingo-1, and we optimized the transduction of neural stem/progenitor cells using lentiviral vectors encoding Lingo-1 shRNA. The addition of DAPT decreased the expression of Notch intracellular domain (NICD) as well as the downstream genes Hes1 and Hes5. Expression of NeuN, CNPase and GFAP in DAPT treated cells and expression of NeuN in Lingo-1 shRNA treated cells confirmed differentiation of neural stem/progenitor cells into neurons, oligodendrocytes and astrocytes. These results revealed that while Lingo-1 shRNA and Notch signaling inhibitor DAPT both promoted differentiation of neural stem cells into neurons, only DAPT was capable of driving neural stem/progenitor cells differentiation into oligodendrocytes and astrocytes. Since we were able to show that both Lingo-1 shRNA and DAPT could drive neural stem/progenitor cells differentiation, our data might aid the development of more effective SCI therapies using Lingo-1 shRNA and DAPT.

Introduction

Spinal cord injury (SCI), a serious injury of the central nervous system (CNS) caused by trauma or disease, often results in the loss of nervous tissue and subsequently leads to the loss of motor and sensory function (Nandoe Tewarie et al., 2009). In recent years, the incidence of CNS diseases has greatly increased with the development of our economy, the prevalence of private car ownership and high-altitude operations. Patients with CNS diseases not only suffer physical and psychological damage, but also place large economic burdens on family and society. At present, CNS injuries, especially SCI prevention, diagnosis, treatment and rehabilitation have become major worldwide challenges. While traditional surgical and hormone therapy for SCI are commonly used, they are unable to remedy the various secondary neurological dysfunctions after injury. Therefore, focus on SCI treatment should be to restore nerve function by promoting axon regeneration and reconnecting severed axons with their original targets (Campos et al., 2004).

Due to nature of CNS injuries, regenerating damaged nerves and recovering nerve function represents a significant challenge. Mechanical damage and the presence of inflammatory factors develop massive cell death, and render the injury site unable to support the growth of new cells. Myelin associated inhibitory factors such as oligodendrocyte myelin glycoprotein (OMgp), myelin-associated glycoprotein (MAG), and neurite outgrowth inhibitory A (Nogo A) interfere with nerve regeneration and hinder the recovery process (Fitch and Silver, 2008, McDonald and Sadowsky, 2002). Accordingly, stem/progenitor cell transplantation and antagonists of myelin associated inhibitory factors have become potential treatments (Gil et al., 2009).

Leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1 (Lingo-1) is a transmembrane protein selectively expressed in neurons and oligodendrocytes of the brain and spinal cord. It combines with OMgp, MAG and Nogo A to form complex, which activates RhoA, a kinase of the Ras family, to inhibit CNS axon sprouting and myelination (Lee et al., 2007; Mi et al., 2004, Mi et al., 2008). Inhibition of Lingo-1 or inactivation of lingo-1 contributes to the differentiation of oligodendrocytes in vivo and in vitro. In addition, inhibition of Lingo-1promotes myelination and maintains axon integrity in vivo (Jepson et al., 2012, Mi et al., 2005b).

Massive cell death at the site of injury aggravates sustained changes of the microenvironment and hinders recovery. The transplantation of exogenous cells can act as a bridge over the damaged area and stimulate injury repair. Our previous studies suggested that stem cells could promote neuronal survival, axonal growth and nerve regeneration in spinal cord injury and ischemia brain diseases (Bin Deng et al., 2010, Deng et al., 2008). When transplanted into SCI mice, neural stem/progenitor cells can promote phagocytosis and mitigate secondary injury caused by the inflammatory response after SCI (Cusimano et al., 2012). Additionally, neural stem/progenitor cells have the ability to self-renew and differentiate into multiple cell types. When used to treat CNS diseases, they may be able to repair the site of injury by producing growth factors to change the microenvironment, and to play an important role in neuroprotection by differentiating into neurons and glial cells (Sandner et al., 2012). N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-Phenyl -glycinet-butylester (DAPT) is an effective inhibitor of the Notch signaling pathway, which delivers cell signals through receptors and ligands of adjacent cells and influences the morphology and neural differentiation of NSCs (Kageyama et al., 2008, Kanungo et al., 2008, Nasoohi et al., 2012, Pierfelice et al., 2011). Here, we use lentiviral vectors encoding Lingo-1 shRNA and the Notch signaling inhibitor DAPT to treat rat neural stem/progenitor cells in vitro. Both Lingo-1 shRNA and DAPT promoted differentiation of neural stem/progenitor cells into neurons, which was detected by western blot and immunofluorescence. Our data may provide a basis for the application of Lingo-1shRNA and DAPT to CNS injuries transplantation therapy.

Section snippets

Nestin and Sox-2 expression confirmed cultured cells to be neural stem/progenitor cells

We stained for Nestin and Sox-2 to confirm that the cells we collected from the hippocampus of rat embryos were neural stem/progenitor cells. The cells isolated from the rat embryos formed spheroids and grew continuously. The fringes of these spheroids were bright under the microscope, while the cell centers were darker (Fig. 3D and G). The cells were cultured on slides treated with polylysine. Adherent cells were then stained using the Nestin or Sox-2 primary antibody, a 488/594-conjugated

Optimized viral-mediated transduction of lentiviral vectors encoding Lingo-1 shRNA

Rat neural stem/progenitor cells were incubated with GFP expressing Lingo-1 shRNA lentiviral vectors at different viral concentrations (MOI of 2, 10, 20, 50 and 100). Transduction of GFP expressing lentiviral vectors in neural stem/progenitor cells was detected by flow cytometry after 96 h (Fig. 2A and B). As shown in Fig. 2A, the transduction rate increased from MOI 2 to MOI 100 and similar levels of vector transduction were achieved from MOI 20 to MOI 100. At MOI of 2 and 10, the transduction

Discussion

Regeneration after CNS diseases remains a pressing medical problem. While the CNS has been viewed as non-renewable, the discovery of myelin associated inhibitory factors has offered new possibilities. As a key myelin associated inhibitory factor, Lingo-1 has been found to be a negative regulator of neurodevelopment, axon growth and myelination (Chen et al., 2006, Mi et al., 2005a). In this study, we report a novel feature of Lingo-1 in neural stem/progenitor cells differentiation. Using Lingo-1

Conclusions

In our study, we treated the neural stem/progenitor cells with both Lingo-1 shRNA and the Notch pathway inhibitor, DAPT. We observed that knockdown of the Lingo-1 gene and treatment with DAPT, which increased the expression of Lingo-1, both had a similar effect of promoting NSC differentiation into neurons. Our results suggested that there were more complex signaling factors involved in the regulation of neuronal differentiation, and the mechanistic relationship between the Notch signaling

Lentiviral vectors preparation

Lentiviral vectors were constructed by the GeneChem Company (Shanghai, China) to silence Lingo-1 in rat NSCs. A green fluorescent protein (GFP) gene tag was encoded in the vector sequence, and the oligonucleotides were ligated to HpaI- and XhoI-digested pFU-GW-RNAi (GeneChem, Shanghai, China). Oligonucleotides were designed with the sequences: 5′-TAAGCACAACATCGAAATTGAA TTCAAGAGATTCAATTTCGATGTTGTGCTTTTTTTTC-3′. Negative control shRNA(NC-shRNA) was designed with the oligonucleotide sequence as

Conflict of interest

All authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant nos. 81171711, 81472069 and 81571105) and Natural Science Foundation Projects of Guangdong Province (Grant nos. S2013020012818 and 2015A030313186).

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¹: These authors contributed equally to this work.

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Research ReportLingo-1 shRNA and Notch signaling inhibitor DAPT promote differentiation of neural stem/progenitor cells into neurons

Highlights

Abstract

Introduction

Section snippets

Nestin and Sox-2 expression confirmed cultured cells to be neural stem/progenitor cells

Optimized viral-mediated transduction of lentiviral vectors encoding Lingo-1 shRNA

Discussion

Conclusions

Lentiviral vectors preparation

Conflict of interest

Acknowledgments

Mol. Cell. Neurosci.

Brain Res. Rev.

Cytotherapy

Cell Stem Cell

Exp. Neurol.

FEBS Lett.

J. Biol. Chem.

Lancet

Int. J. Biochem. Cell Biol.

Neuroscience

Neuron

Dev. Biol.

Neuroscience

Neuron

Biomaterials

Intravenously administered BMSCs reduce neuronal apoptosis and promote neuronal proliferation through the release of VEGF after stroke in rats

Neurol. Res.

Research Report
Lingo-1 shRNA and Notch signaling inhibitor DAPT promote differentiation of neural stem/progenitor cells into neurons