Olfactory ensheathing cells facilitate neurite sprouting and outgrowth by secreting high levels of hevin

https://doi.org/10.1016/j.jchemneu.2019.101728Get rights and content

Highlights

  • •

    OECs are good candidates for repairing nervous injuries due to their various properties that promote synaptogenesis.

  • •

    Olfactory ensheathing cells to stimulate neurite sprouting and outgrowth by secreting high levels of hevin.

  • •

    OECs have a greater capacity for promoting synaptic formation than astrocytes.

Abstract

Transplantation of olfactory ensheathing cells (OECs) has been shown to enhance synapse formation. However, the mechanisms underlying this effect are not completely understood. We performed profiling of the OEC and astrocyte secretomes via a proteomics approach, in case hevin secreted by astrocytes might be involved in the formation of synapses. Semi-quantitative proteomic analysis revealed that 25 proteins were highly expressed, and 22 were weakly expressed in OEC conditioned medium compared with astrocyte conditioned medium. These molecules are highly associated with neural differentiation and regeneration, enzyme regulatory activity, and growth factor binding. The quantification data of clusterin, fibronectin, hevin, insulin-like growth factor binding protein 2 and secreted protein acidic and rich in cysteine were further confirmed by western blotting. Moreover, the addition of hevin in the culture medium improved neurite sprouting and outgrowth of differentiated neural stem cells. The greater expression of hevin in OEC conditioned medium than in astrocyte conditioned medium was associated with a greater capacity of synaptic formation. Thus, our results indicate that soluble factors secreted by OECs provide a permissive environment for nerve repair, and hevin is one of the key molecules facilitating neurite sprouting and outgrowth.

Introduction

Olfactory ensheathing cells (OECs), also known as olfactory ensheathing glia cells, are a unique population of macroglia that are found both centrally and peripherally along the olfactory nerve (Lipson et al., 2003), and display some properties shared by both astrocytes and Schwann cells. These cells exhibit robust neurite extension and functional recovery after transplantation (Boruch et al., 2001; Liu et al., 2010; Shi et al., 2010; Duan et al., 2011; Sethi et al., 2014). OECs can guide injured axons back into the intact central nervous system, as demonstrated in animals with spinal cord injuries (SCI) (Tabakow et al., 2013). Direct OEC-neurite cell contact can provide a permissive substrate that can overcome the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core (Barnett, 2004). In addition, OECs secrete large amounts of neurotrophic factors and adhesion molecules that promote neurite outgrowth and neuron regeneration (Barnett and Riddell, 2004;Liu et al., 2010), and reactive astrocytes secrete molecules that inhibit axon growth, such as chondroitin sulfate proteoglycans (Bampton et al., 2005). Several trials using OECs as a treatment in SCI models and even human patients with SCI (Féron et al., 2005; Mackay-Sim et al., 2008; Zhang et al., 2017) have yielded encouraging results. However, most of these studies have focused on the evaluation of functional recovery, and the details of paracrine and neurite outgrowth involved in the OEC mediated tissue repair process remains largely unknown.

Hevin belongs to the SPARC (Johnston et al., 1990; Brekken and Sage, 2000 2001) family of matricellular proteins, which have many functions. Hevin is widely expressed in the central nervous system (CNS) and may be involved in the development of the CNS and the formation of synapses (Mendis and Brown, 1994;Mendis et al., 1994, 1995, 1996;Lively et al., 2007). Moreover, Hakan Kucukdereli al. (Kucukdereli et al., 2011) have found that hevin secreted by astrocytes contributes to the regulation of synaptogenesis between cultured rat retinal ganglion cells.

The aim of the present study was to investigate whether OECs might also secrete proteins that modulate synapse formation and to identify the differences in hevin expression between OECs and astrocytes. We performed profiling of OEC and astrocyte secretomes via a proteomics approach. Through semi-quantitative secretome analysis, we found that some proteins had higher expression in OEC conditioned medium than astrocyte conditioned medium, and the differential expression levels of secreted proteins were further confirmed by western blotting. Compared with astrocytes, OECs facilitated more neurite sprouting and outgrowth may shed light on the regulatory mechanism of OECs in nerve repair.

Section snippets

Animals

Fetal Sprague-Dawley (SD) rats obtained from the Laboratory Animal department of Central South University were used in the study. This study was carried out in strict accordance with the guidelines established by the Committee on the Use and Care of Animals of Hunan Province, P. R. China. The protocol was approved by the Ethics Committee of the University of Hunan Normal University (permit number: 010,008). All possible effort was made to minimize suffering. All experiments were approved by and

Culture and identification of OECs and astrocytes

We followed protocols for the generation of OEC and astrocyte cultures and successfully derived cells. Microscopy observations indicated that the OECs grew vigorously, and most cells showed a bipolar or multipolar shape with spindle morphology (Fig.1A), whereas the astrocytes exhibited heterogeneous morphologies with multiple thin processes (Fig.1D). After passaging, the purified cells were immunopositive for P75 and GFAP, characteristic markers of OECs and astrocytes, respectively (Fig.1B,E).

Discussion

Recent studies have suggested that OECs are an attractive candidate for neurological repair(Huang et al., 2003; Ge et al., 2016). Our previous studies have also demonstrated that OECs secrete a variety of proteins (Liu et al., 2010). Some of these proteins have been shown to induce and promote the differentiation of stem cells into neurons (Duan et al., 2011; Zeng et al., 2013), such as brain-derived neurotrophic factor, basic fibroblast growth factor and nerve growth factor. However, the

Conclusion

In summary, we performed secretome profiling between OECs and astrocytes and quantified the significantly differentially expressed proteins. The results demonstrated that hevin secreted by OECs facilitates neurite sprouting and outgrowth. On the basis of the overexpression of hevin in OCM, OECs have a greater capacity for synaptic formation than astrocytes. However, further experiments will be necessary to confirm the ways in which hevin contributes to synapse formation. The in vitro data may

Author’s contribution statement

L.G. Y.Z. and P.W. were responsible for study design, mass spectrum, data collection and analysis, Western blot and contributed to the drafting of the article. D.D. and Y.S.L. X.H.T. was responsible for cell culture, immunofluorescence analysis and contributed to drafting of the article. L.Y.Q. was responsible for Western blot. L.G was responsible for study design and contributed to the drafting of the article. M.L. and P.C. were responsible for study design and analysis, contributed to the

Funding

This work was supported by the National Natural Science Foundation of China (31670838, 31370817), the Scientific Research Fund of the Hunan Provincial Education Department (16B159), and the Hunan Provincial Innovation Foundation for Postgraduates (NO.CX2018B309, NO.CX2018B240).

Ethical statement

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Second Affiliated Hospital of Hunan Normal University(NO.2016049). All surgery was performed under anesthesia, and all efforts were made to minimize suffering.

Informed consent

Not applicable.

Declaration of Competing Interest

The authors have no conflicts of interest to disclose

Acknowledgements

We are grateful to Dr. Ying Wang for advice and helpful discussion.

References (40)

  • X. Shi et al.

    A long-term observation of olfactory ensheathing cells transplantation to repair white matter and functional recovery in a focal ischemia model in rat

    Brain Res.

    (2010)
  • E. Au et al.

    SPARC from olfactory ensheathing cells stimulates Schwann cells to promote neurite outgrowth and enhances spinal cord repair

    J. Neurosci.

    (2007)
  • E.T. Bampton et al.

    Osteonectin is a Schwann cell-secreted factor that promotes retinal ganglion cell survival and process outgrowth

    Eur. J. Neurosci.

    (2005)
  • S.C. Barnett

    Olfactory ensheathing cells: unique glial cell types

    J. Neurotrauma

    (2004)
  • S.C. Barnett et al.

    Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats

    J. Anat.

    (2004)
  • A.V. Boruch et al.

    Neurotrophic and migratory properties of an olfactory ensheathing cell line

    Glia.

    (2001)
  • J.G. Boyd et al.

    Proteomic evaluation reveals that olfactory ensheathing cells but not Schwann cells express calponin

    Glia.

    (2006)
  • R.A. Brekken et al.

    SPARC, a matricellular protein: at the crossroads of cell-matrix communication

    Matrix Biol.

    (2001)
  • R.A. Brekken et al.

    Expression and characterization of murine hevin (SC1), a member of the SPARC family of matricellular proteins

    J. Histochem. Cytochem.

    (2004)
  • R. Cao et al.

    Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

    J. Proteome Res.

    (2006)

    • Cited by (0)

    1

    These authors contributed equally to this work.

    View full text
    © 2019 Published by Elsevier B.V.