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. 2018 Nov 6;8(1):16443.
doi: 10.1038/s41598-018-34585-3.

VGF Nerve Growth Factor Inducible Is Involved in Retinal Ganglion Cells Death Induced by Optic Nerve Crush

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Free PMC article

VGF Nerve Growth Factor Inducible Is Involved in Retinal Ganglion Cells Death Induced by Optic Nerve Crush

Hiroto Takeuchi et al. Sci Rep. .
Free PMC article

Abstract

VGF nerve growth factor inducible (VGF) is a polypeptide that is induced by neurotrophic factors and is involved in neurite growth and neuroprotection. The mRNA of the Vgf gene has been detected in the adult rat retina, however the roles played by VGF in the retina are still undetermined. Thus, the purpose of this study was to determine the effects of VGF on the retinal ganglion cells (RGCs) of mice in the optic nerve crush (ONC) model, rat-derived primary cultured RGCs and human induced pluripotent stem cells (iPSCs)-derived RGCs. The mRNA and protein of Vgf were upregulated after the ONC. Immunostaining showed that the VGF was located in glial cells including Müller glia and astrocytes but not in the retinal neurons and their axons. AQEE-30, a VGF peptide, suppressed the loss of RGCs induced by the ONC, and it increased survival rat-derived RGCs and promoted the outgrowth of neurites of rat and human iPSCs derived RGCs in vitro. These findings indicate that VGF plays important roles in neuronal degeneration and has protective effects against the ONC on RGCs. Thus, VGF should be considered as a treatment of RGCs degeneration.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression of the mRNA of the Vgf gene and VGF protein in the optic nerve crush (ONC) model. (A,B) The quantitative data of the level of expression of Vgf mRNA and VGF protein after the ONC are shown. qRT-PCR was used to determine the level of expression of Vgf mRNA. The mRNA of Vgf is increased significantly at 2, 3 and 5 days after the ONC. Western blotting was used to determine the level of expression of VGF protein. The protein of VGF is increased significantly at 7 days after the ONC. Data are the means ± standard error of the means (SEMs). (A: n = 5–7, B: n = 7–12). *P < 0.05, **P < 0.01 versus sham group (A: Dunnett’s test, B: Student’s t-tests). (C) Representative images of immunostained retinal sections to determine the site of VGF protein expression after the ONC. Scale bar = 50 μm. The measurements of the intensity of the (D) whole retina, (E) retinal nerve fiber layer plus ganglion cell layer (RNFL + GCL), and (F) inner plexiform layer (IPL) were performed. The VGF protein increased significantly in all areas 7 days after the ONC. Data are the means ± SEMs. (n = 4 or 5). *P < 0.05 versus sham group (Student’s t-tests). In this figure, the cropped blots are used. The full-length blots are showed in Supplementary Fig. 1.
Figure 2
Figure 2
Retinal location of VGF in the sham group and 7 days after ONC group. (A,B) VGF expression (red) was co-localized with GS+ Müller glia (green) and partially co-localized with GFAP+ astrocytes (green) in both group. (C) VGF expression (red) was localized around the NF-H+ nerve fiber (green). Scale bar = 25 μm.
Figure 3
Figure 3
VGF expression and localization in optic nerve. (A) Representative images showing the sites of the VGF expression in the optic nerve. The intensity of VGF expression was measured anterior to the crush site (B), the crush site (C), and posterior to the crush site (D). The expression of VGF is significantly higher on 1, 2, 5, and 7 days after the ONC at all 3 areas. (E) VGF expression (red) was co-localization with GFAP+ astrocytes (green) in both groups. Data are the means ± SEMs (n = 5). *P < 0.05, **P < 0.01 versus sham group (Dunnett’s test). Scale bar = 50 μm.
Figure 4
Figure 4
AQEE-30, a VGF peptide, suppresses the loss of retinal ganglion cells (RGCs), but do not change the number of microglia induced by ONC. (A) Representative images showing fluorogold-labeled RGCs in flat-mounted retinas at 10 days after the ONC. (B) The number of surviving RGCs was counted at the central, middle, and peripheral areas. (C) The total number of surviving RGCs is shown. AQEE-30 inhibited the loss of RGCs. Data are the means ± SEMs. (n = 4–12). **P < 0.01 versus sham group (Student’s t-test). #P < 0.05, ##P < 0.01 versus vehicle-treated group (Student’s t-test). Scale bar = 50 μm. (D) The number of FG-labeled microglia was counted at the central, middle, and peripheral areas. (E) The total number of FG-labeled microglia is shown. AQEE-30 and Brimonidine did not change the total number of FG-labeled microglia. **P < 0.01 versus sham group (Student’s t-test).
Figure 5
Figure 5
VGF Overexpression of VGF does not inhibit the death of RGCs induced by ONC. (A) The expression levels of VGF in the VGF-overexpressing mice (Tg) retina was higher than wildtype mice (WT) retina. Data are the means ± SEMs. (n = 3). *P < 0.05 versus WT group (Student’s t-test). (B) Representative images of normal retinal structure in WT mice and VGF-overexpressing mice. (C) Quantitative data of retinal thickness in normal retina. In each layer, the thickness was not significantly different between WT mice and VGF-overexpressing mice. (D) The number of cells in GCL is not significantly different between WT mice and VGF-overexpressing mice. Data are the means ± SEMs. (n = 9–11). Scale bar = 50 μm. (E) Representative fluorescence images showing RGCs in the flat-mounted retina at 10 days after the ONC. (F) Quantitative data showing the number of fluorogold-labeled RGCs at the central, middle, and peripheral areas. (G) Total number of surviving RGCs was measured. Overexpression of VGF did not affect the death of RGCs. Data are the means ± SEMs. (n = 5–10). Scale bar = 50 μm. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear layer, OPL; outer plexiform layer, ONL; outer nuclear layer. The cropped blots are used in this figure. The full-length blots are presented in Supplementary Fig. 2.
Figure 6
Figure 6
AQEE-30 promotes neurites outgrowth from RGCs in vitro. (A) Typical images of cultured rat-derived RGCs at 3 days after purification. The neurites are stained green by Calcein-AM+ (green) and cells by Tuj-1+ (red). (B) Quantitative data showing the neurites length of RGCs. AQEE-30 at 1 µM promotes neurites outgrowth in rats-derived RGCs. Data are the means ± SEMs (n = 6). *P < 0.05 versus control group (Student’s t-test). Scale bar = 50 μm. (C) Quantitative data showing the Survival RGCs. (D) Immunostained images of human induced pluripotent stem cells (iPSCs) derived RGCs culture at 50 days after the neuronal induction. The neurites of the cells are stained green by Tuj-1+. (E) AQEE-30 at 1 µM to 10 µM also promotes neurite outgrowth in human iPSCs-derived RGCs in a concentration dependent manner. Data are the means ± SEMs. (n = 4). **P < 0.01 versus control group (Dunnett’s test).

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