Volume 12, Issue 2 (March & April 2021)
BCN 2021, 12(2): 223-232 |
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Nedaei K, Hesaraki M, Mazloomzadeh S, Totonchi M, Biglari A R. Lentiviral Mediated Expression of Soluble Neuropilin 1 Inhibits Semaphorin 3A-mediated Collapse Activity in Vitro. BCN 2021; 12 (2) :223-232
URL: http://bcn.iums.ac.ir/article-1-1405-en.html
URL: http://bcn.iums.ac.ir/article-1-1405-en.html
1- Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
2- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
3- Social Determinants of Health Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
4- Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
2- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
3- Social Determinants of Health Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
4- Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
Abstract:
Introduction: Semaphorin 3A (Sema 3A) is a secreted protein, which plays an integral part in developing the nervous system. It has collapse activity on the growth cone of dorsal root ganglia. After the development of the nervous system, Sema 3A expression decreases. Neuropilin 1 is a membrane receptor of Sema 3A. When semaphorin binds to neuropilin 1, the recruitment of oligodendrocyte precursor cells to the demyelinated site decreases. In Multiple Sclerosis (MS), Sema 3A expression increases and inhibits oligodendrocyte precursor cell differentiation. Therefore, the remyelination of axons gets impaired. We hypothesized that the function of Sema 3A could be inhibited by neutralizing its binding to membrane NRP1.
Methods: we cloned a soluble form of mouse Neuropilin 1 (msNRP1) in a lentiviral vector and expressed the recombinant protein in HEK293T cells. Then, the conditioned medium of the transduced cells was used to evaluate the effects of the msNRP1 on the inhibition of Sema 3A-induced growth cone collapse activity. Dorsal root ganglion explants of timed pregnant (E13) mice were prepared. Then, the growth cone collapse activity of Sema 3A was assessed in the presence and absence of msNRP1-containing conditioned media of transduced and non-transduced HEK293T cells. Comparisons between groups were performed by 1-way ANOVA and post hoc Tukey tests.
Results: msNRP1 was successfully cloned and transduced in HEK293T cells. The supernatant of transduced cells was concentrated and evaluated for the production of msNRP1. ELISA results indicated that transduced cells secreted msNRP1. Growth cone collapse assay showed that Sema 3A activity was significantly reduced in the presence of the conditioned medium of msNRP1-transduced HEK293T cells. Conversely, a conditioned medium of non-transduced HEK293T cells could not effectively prevent Sema 3A growth cone collapse activity.
Conclusion: Our results indicated that msNRP1 was successfully produced in HEK293T cells. The secreted msNRP1 effectively prevented Sema 3A collapse activity. Therefore, msNRP1 can increase remyelination in MS lesions, although more studies using animal models are required.
Methods: we cloned a soluble form of mouse Neuropilin 1 (msNRP1) in a lentiviral vector and expressed the recombinant protein in HEK293T cells. Then, the conditioned medium of the transduced cells was used to evaluate the effects of the msNRP1 on the inhibition of Sema 3A-induced growth cone collapse activity. Dorsal root ganglion explants of timed pregnant (E13) mice were prepared. Then, the growth cone collapse activity of Sema 3A was assessed in the presence and absence of msNRP1-containing conditioned media of transduced and non-transduced HEK293T cells. Comparisons between groups were performed by 1-way ANOVA and post hoc Tukey tests.
Results: msNRP1 was successfully cloned and transduced in HEK293T cells. The supernatant of transduced cells was concentrated and evaluated for the production of msNRP1. ELISA results indicated that transduced cells secreted msNRP1. Growth cone collapse assay showed that Sema 3A activity was significantly reduced in the presence of the conditioned medium of msNRP1-transduced HEK293T cells. Conversely, a conditioned medium of non-transduced HEK293T cells could not effectively prevent Sema 3A growth cone collapse activity.
Conclusion: Our results indicated that msNRP1 was successfully produced in HEK293T cells. The secreted msNRP1 effectively prevented Sema 3A collapse activity. Therefore, msNRP1 can increase remyelination in MS lesions, although more studies using animal models are required.
Type of Study: Original |
Subject:
Cellular and molecular Neuroscience
Received: 2020/01/15 | Accepted: 2020/08/2 | Published: 2021/03/1
Received: 2020/01/15 | Accepted: 2020/08/2 | Published: 2021/03/1
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