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Inhibition of Retinal Ganglion Cell Axonal Outgrowth Through the Amino-Nogo-A Signaling Pathway

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Abstract

Nogo-A is a myelin-derived inhibitor playing a pivotal role in the prevention of axonal regeneration. A functional domain of Nogo-A, Amino-Nogo, exerts an inhibitory effect on axonal regeneration, although the mechanism is unclear. The present study investigated the role of the Amino-Nogo–integrin signaling pathway in primary retinal ganglion cells (RGCs) with respect to axonal outgrowth, which is required for axonal regeneration. Immunohistochemistry showed that integrin αv, integrin α5 and FAK were widely expressed in the visual system. Thy-1 and GAP-43 immunofluorescence showed that axonal outgrowth of RGCs was promoted by Nogo-A siRNA and a peptide antagonist of the Nogo-66 functional domain of Nogo-A (Nep1–40), and inhibited by a recombinant rat Nogo-A-Fc chimeric protein (△20). Western blotting revealed increased integrin αv and p-FAK expression in Nogo-A siRNA group, decreased integrin αv expression in △20 group and decreased p-FAK expression in Nep1–40 group. Integrin α5 expression was not changed in any group. RhoA G-LISA showed that RhoA activation was inhibited by Nogo-A siRNA and △20, but increased by Nep1–40 treatment. These results suggest that Amino-Nogo inhibits RGC axonal outgrowth primarily through the integrin αv signaling pathway.

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Abbreviations

RGC:

Retinal ganglion cell

CNS:

Central nervous system

siRNA:

Small interfering RNA

FAK:

Focal adhesion kinase

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

ECL:

Enhanced chemiluminescence

SD:

Sprague–Dawley

PBS:

Phosphate buffered saline

NC:

Negative control

HRP:

Horseradish peroxidase

GCL:

Ganglion cell layer

INL:

Inner nuclear layer

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Acknowledgments

This study was supported by the National Nature Science Foundation of China (No. 31070968) and the National Natural Science Foundation of China (No. 81000378).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of ophthalmology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China

    Yan Huo, Shu-Xing Ji, Huan Zou, Min Lang, Zheng Zheng, Xiao-Feng Cai, Wei Liu, Chun-Lin Chen & Jian Ye

  2. Department of ophthalmology, 305 Hospital of PLA, Beijing, China

    Xiao-Lei Yin

  3. Department of Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China

    Yuan-Guo Zhou

  4. Department of ophthalmology, Xinqiao Hospital, Third Military Medical University, Chongqing, China

    Rong-Di Yuan

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  1. Yan Huo
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Correspondence to Rong-Di Yuan or Jian Ye.

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11064_2013_1032_MOESM1_ESM.jpg

Identification of primary cultured retinal ganglion cells. Immunofluorescence staining was performed using anti-Thy-1 (green). DAPI-counterstained nuclei are shown in blue. Scale bar: 50 μm. (JPEG 151 kb)

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Huo, Y., Yin, XL., Ji, SX. et al. Inhibition of Retinal Ganglion Cell Axonal Outgrowth Through the Amino-Nogo-A Signaling Pathway. Neurochem Res 38, 1365–1374 (2013). https://doi.org/10.1007/s11064-013-1032-1

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  • DOI: https://doi.org/10.1007/s11064-013-1032-1

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