Fig 1: GPNMB mediates RGC survival and axon regeneration.(A) Relative Slc6a4 mRNA expression in the VT retina two weeks after infection of AAV2-shSlc6a4 virus compared to AAV2-shControl (AAV2-shCtr) virus as a control (n = 4 mice/condition, two-tailed unpaired t-test). (B-C) In in DBA/2J-Gpnmb+ mice, peripheral VT RGCs are protective by AAV2-shSlc6a4 virus compared to AAV2-shCtr virus two weeks after ONC, while in DBA/2J mice (which carry mutations in Gpnmb gene), VT RGCs are not protected by AAV2-shSlc6a4 virus. However, Peripheral VT RGCs following AAV2-GFP-Gpnmb virus infection into the ventral retina of DBA/2J mice are protective after injury (n = 5 mice/condition, one-way ANOVA). (D) CTB-labeled regenerated axons beyond the injury site (*) are observed in DBA/2J-Gpnmb+ mice after AAV2-shSlc6a4 virus infection or DBA/2J mice after AAV2-GFP-Gpnmb virus infection into the ventral retina. However, DBA/2J-Gpnmb+ mice carrying AAV2-shCtr virus or DBA/2J mice carrying AAV2-shSlc6a4 virus show little or minimal axon regeneration two weeks after ONC, respectively. (E-F) Quantitative analysis of axon regeneration in DBA/2J-Gpnmb+ or DBA/2J mice having AAV2-shSlc6a4, AAV2-shCtr or AAV2-GFP-Gpnmb virus in the ventral retina two weeks after ONC. The average of the distance of the CTB-labeled five longest axons from the lesion site (E) and the number of regenerated axons at 400 and 800 μm from the lesion site are shown (F). (n = 5 mice /condition, one-way-ANOVA (E), two-way ANOVA (F)). Data presented as mean ± SD. N.S., not significant; Scale bars represent 20 μm (B) and 100 μm (D).
Fig 2: Loss of SERT leads to neuroprotection of peripheral VT RGCs after optic nerve injury.(A) Schema of RGC survival analysis in the retina five days or two weeks after ONC in WT and Slc6a4-/- mice. (B) Five days after ONC, WT VT RGCs are markedly reduced, while ~80% of VT RGCs in Slc6a4-/- mice still survive (n = 5 mice/condition, two-way ANOVA). (C-D) Two weeks after ONC, VT RGCs in Slc6a4-/- mice are more resistant to injury compared to RGCs in other regions in Slc6a4-/- mice or all regions in WT mice (n = 5 mice/condition, two-way ANOVA). (E) Four weeks after ONC, peripheral, but not middle VT RGCs in Slc6a4-/- mice are more protective compared to those in WT mice (n = 4 mice/condition, two-way ANOVA). (F) Fluoxetine, a serotonin reuptake inhibitor has no neuroprotective effects in VT and DN RGCs two weeks after ONC (n = 4 mice/condition, two-way ANOVA). Data presented as mean ± SD. N.S., not significant; Scale bar represents 20 μm.
Fig 3: TLR9 activation by ODN inhibits SERT function and expression. a. 5-HT uptake was measured after 6-min incubation of 5-HT 0.2 μM. ODN concentrations assayed were 1 and 5 μg/ml. The stimulation periods were 30 min (short-term) and 1 day (long-term). The results are expressed as the percentage of the control (100%) and are the mean plus SD of seven independent experiments. Absolute control values were 7.77 ± 0.74 and 8.72 ± 0.68 pmol 5-HT/mg protein at 30 min and 1 day, respectively. b. Real-time PCR analysis of SERT mRNA expression level in cells treated for 30 min and 1 day with ODN 5 μg/ml. Relative quantification was performed with the comparative Ct method (2.–ΔΔCt) normalized by HPRT1 and GAPDH mean. Results are expressed as arbitrary units (control = 1) and are the mean plus SD of six independent experiments. c. Immunodetection and quantification of SERT protein expression by western blot in cell lysate and apical membrane from Caco-2/TC7 cells treated with ODN 1 and 5 μg/ml for 1 day using β-actin as an internal control of the protein load (SERT/β-actin ratio). The results are expressed as percentage of the control value and are the mean plus SD of four independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control value
Fig 4: TLR9 located in cell membrane is involved in SERT inhibition. Caco-2/TC7 cells were treated for 1 day with ODN 5 μg/ml with or without a 1-h pre-treatment with chloroquine (CQ) 20 μM or TLR9 antibody (TLR9 Ab) 1:100. a. Uptake of 5-HT was measured after 6 min of incubation with 5-HT 0.2 μM. The results are expressed as the percentage of the control (100%). Results are the mean plus SD of four independent experiments. Absolute control value was 1.584 ± 0.35 pmol 5-HT/mg protein. b. 5-HT apical-to-basal fluxes. 5-HT was used at 0.1 μM, and samples were taken every 10 min from the basal side. The results are expressed as the percentage of the control value (100%) and are the mean plus SD of five independent experiments. Absolute control values in pmol 5 HT/10 min were 0.31 ± 0.04. c. Real-time PCR analysis of TLR9 mRNA expression in Caco-2/TC7 cells treated during 1 day with ODN 5 μg/ml. Relative quantification was performed in triplicate with the comparative Ct method (2–ΔΔCt) normalized by HPRT1 and GAPDH mean. Results are expressed as arbitrary units (control = 1) and are the mean plus SD of three independent experiments. d. Immunodetection and quantification of TLR9 protein expression by western blot in cell lysate and apical membrane from Caco-2/TC7 cells treated with ODN 5 μg/ml for 1 day. Results are expressed as SERT/β-actin ratio and are the mean plus SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control value, unless otherwise indicated; #P < 0.05 and.##P < 0.01 compared with ODN-treated cells
Fig 5: VT RGC death occurs after SERT expression on VT axons is elevated following optic nerve injury.(A) Ipsilateral RGCs (ZsGreen-SERT+ RGCs) are located in the peripheral VT, but not DN retina of Slc6a4::Cre;R26RZsGreen mice. (B) Ipsilateral RGCs (ZsGreen-SERT+ RGCs) are more predominantly located in the peripheral (0–200 or 200–400 μm) compared to the central (400–600 μm) VT retina in Slc6a4::Cre;R26RZsGreen mice (n = 5 mice, one-way ANOVA). (C-D) Time-course of ipsilateral RGC (ZsGreen-SERT+ cells) death in the peripheral VT retina after ONC (n = 5 mice/condition, one-way ANOVA). (E) In the peripheral VT retina four days after ONC in Slc6a4::Cre;R26RZsGreen mice, ipsilaterally-projecting RGCs (ZsGreen-SERT+/βIII-tubulin+) are more vulnerable to injury than contralaterally-projecting RGCs (ZsGreen-SERT-negative/βIII-tubulin+). (F) Quantitative analysis of ipsilateral (ZsGreen-SERT+/βIII-tubulin+) and contralateral (ZsGreen-SERT-negative/βIII-tubulin+) RGC survival (%) in the peripheral VT retina four days after ONC (n = 5 mice, two-tailed unpaired t-test). (G-H) In Slc6a4::Cre;R26RZsGreen mice, ZsGreen-SERT expression (Slc6a4::Cre-derived ZsGreen expression) is upregulated on VT axons (Axon) one and three days after ONC (n = 4 mice/condition, one-way ANOVA). (I) Endogenous SERT expression is upregulated on injured VT axons of WT mice one day after ONC (arrows) compared to the intact VT axons. SERT expression is not observed on injured VT axons of Slc6a4-/- mice. (J) Quantitative analysis of SERT expression changes in axons of VT retina one day after ONC compared to the intact counterparts (n = 4 mice, two-tailed unpaired t-test). Data presented as mean ± SD. N.S., not significant; Scale bars represent 20 μm (A (high magnifications), B, C, E, G, I) and 200 μm (A (whole retina)).
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