Activating Transcription Factor 3 (ATF3) Protects Retinal Ganglion Cells and Promotes Functional Preservation After Optic Nerve Crush | IOVS (original) (raw)

February 2020

Volume 61, Issue 2

Figure 1.

ONC leads to preferential elevation of ATF3 level in αRGC. (A) Transverse sections of intact uninjured retina and retina 10 dpc were stained for OPN (red), ATF3 (green) and RBPMS (gray scale). (B) Merged images for ATF3/RBPMS and ATF3/OPN staining at 10 dpc. (C) Percent of αRGCs and non-αRGCs that survived at 10 dpc. (D) Fraction of all RGCs comprised by αRGCs and non-αRGCs in intact retina and at 10 dpc. (E) Percent of ATF3 positive αRGCs and non-αRGCs at 10 dpc. Six and five biological replicates were used for 10 dpc and control, non-crushed eyes groups, respectively. Data are shown as mean ± SEM. Unpaired t-test was used for statistical analysis; P values are shown above corresponding columns.

ONC leads to preferential elevation of ATF3 level in αRGC. (A) Transverse sections of intact uninjured retina and retina 10 dpc were stained for OPN (red), ATF3 (green) and RBPMS (gray scale). (B) Merged images for ATF3/RBPMS and ATF3/OPN staining at 10 dpc. (C) Percent of αRGCs and non-αRGCs that survived at 10 dpc. (D) Fraction of all RGCs comprised by αRGCs and non-αRGCs in intact retina and at 10 dpc. (E) Percent of ATF3 positive αRGCs and non-αRGCs at 10 dpc. Six and five biological replicates were used for 10 dpc and control, non-crushed eyes groups, respectively. Data are shown as mean ± SEM. Unpaired _t_-test was used for statistical analysis; P values are shown above corresponding columns.

Figure 1.

ONC leads to preferential elevation of ATF3 level in αRGC. (A) Transverse sections of intact uninjured retina and retina 10 dpc were stained for OPN (red), ATF3 (green) and RBPMS (gray scale). (B) Merged images for ATF3/RBPMS and ATF3/OPN staining at 10 dpc. (C) Percent of αRGCs and non-αRGCs that survived at 10 dpc. (D) Fraction of all RGCs comprised by αRGCs and non-αRGCs in intact retina and at 10 dpc. (E) Percent of ATF3 positive αRGCs and non-αRGCs at 10 dpc. Six and five biological replicates were used for 10 dpc and control, non-crushed eyes groups, respectively. Data are shown as mean ± SEM. Unpaired _t_-test was used for statistical analysis; P values are shown above corresponding columns.

ONC leads to preferential elevation of ATF3 level in αRGC. (A) Transverse sections of intact uninjured retina and retina 10 dpc were stained for OPN (red), ATF3 (green) and RBPMS (gray scale). (B) Merged images for ATF3/RBPMS and ATF3/OPN staining at 10 dpc. (C) Percent of αRGCs and non-αRGCs that survived at 10 dpc. (D) Fraction of all RGCs comprised by αRGCs and non-αRGCs in intact retina and at 10 dpc. (E) Percent of ATF3 positive αRGCs and non-αRGCs at 10 dpc. Six and five biological replicates were used for 10 dpc and control, non-crushed eyes groups, respectively. Data are shown as mean ± SEM. Unpaired t-test was used for statistical analysis; P values are shown above corresponding columns.

Figure 2.

Effects of rAAV-ATF3 overexpression on RGC survival. (A) Timeline of in vivo study. (B) Scheme of RGC counting; squares represent counting fields. (C) Representative images of regions within whole-mount retinas for different groups. Right image (intact) corresponds to noninjected/non-ONC retina. (D) Quantification of RGCs for different groups. (E) Percentage of surviving RGCs 14 dpc normalized to intact retinas (100%). Eight biological replicates were used for rAAV-ATF3 and rAAV-PLAP groups, three biological replicates were used for intact retina. Each biological replicate represents the average of 12 different fields in the retina. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey post hoc test, was used for D, and unpaired t-test comparison for E. P values are shown above corresponding the graph.

Effects of rAAV-ATF3 overexpression on RGC survival. (A) Timeline of in vivo study. (B) Scheme of RGC counting; squares represent counting fields. (C) Representative images of regions within whole-mount retinas for different groups. Right image (intact) corresponds to noninjected/non-ONC retina. (D) Quantification of RGCs for different groups. (E) Percentage of surviving RGCs 14 dpc normalized to intact retinas (100%). Eight biological replicates were used for rAAV-ATF3 and rAAV-PLAP groups, three biological replicates were used for intact retina. Each biological replicate represents the average of 12 different fields in the retina. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey post hoc test, was used for D, and unpaired _t_-test comparison for E. P values are shown above corresponding the graph.

Figure 2.

Effects of rAAV-ATF3 overexpression on RGC survival. (A) Timeline of in vivo study. (B) Scheme of RGC counting; squares represent counting fields. (C) Representative images of regions within whole-mount retinas for different groups. Right image (intact) corresponds to noninjected/non-ONC retina. (D) Quantification of RGCs for different groups. (E) Percentage of surviving RGCs 14 dpc normalized to intact retinas (100%). Eight biological replicates were used for rAAV-ATF3 and rAAV-PLAP groups, three biological replicates were used for intact retina. Each biological replicate represents the average of 12 different fields in the retina. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey post hoc test, was used for D, and unpaired _t_-test comparison for E. P values are shown above corresponding the graph.

Effects of rAAV-ATF3 overexpression on RGC survival. (A) Timeline of in vivo study. (B) Scheme of RGC counting; squares represent counting fields. (C) Representative images of regions within whole-mount retinas for different groups. Right image (intact) corresponds to noninjected/non-ONC retina. (D) Quantification of RGCs for different groups. (E) Percentage of surviving RGCs 14 dpc normalized to intact retinas (100%). Eight biological replicates were used for rAAV-ATF3 and rAAV-PLAP groups, three biological replicates were used for intact retina. Each biological replicate represents the average of 12 different fields in the retina. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey post hoc test, was used for D, and unpaired t-test comparison for E. P values are shown above corresponding the graph.

Figure 3.

Effects of ATF3 overexpression on RGC axon regeneration after ONC. (A) Representative images of optic nerve sections from rAAV-ATF3- and rAAV-PLAP-treated eyes 10 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods, crush sites are indicated by asterisks. (B) Quantification of regenerating axons as in A. N = 9 and 8 for ATF- and PLAP-injected samples, respectively. Data are shown as mean ± SEM. Two-way ANOVA test was used for B.

Effects of ATF3 overexpression on RGC axon regeneration after ONC. (A) Representative images of optic nerve sections from rAAV-ATF3- and rAAV-PLAP-treated eyes 10 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods, crush sites are indicated by asterisks. (B) Quantification of regenerating axons as in A. N = 9 and 8 for ATF- and PLAP-injected samples, respectively. Data are shown as mean ± SEM. Two-way ANOVA test was used for B.

Figure 3.

Effects of ATF3 overexpression on RGC axon regeneration after ONC. (A) Representative images of optic nerve sections from rAAV-ATF3- and rAAV-PLAP-treated eyes 10 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods, crush sites are indicated by asterisks. (B) Quantification of regenerating axons as in A. N = 9 and 8 for ATF- and PLAP-injected samples, respectively. Data are shown as mean ± SEM. Two-way ANOVA test was used for B.

Effects of ATF3 overexpression on RGC axon regeneration after ONC. (A) Representative images of optic nerve sections from rAAV-ATF3- and rAAV-PLAP-treated eyes 10 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods, crush sites are indicated by asterisks. (B) Quantification of regenerating axons as in A. N = 9 and 8 for ATF- and PLAP-injected samples, respectively. Data are shown as mean ± SEM. Two-way ANOVA test was used for B.

Figure 4.

Effects of ATF3 overexpression on the pSTR and scotopic responses 7 dpc. (A) Representative traces of the observable pSTR at different light intensities. *The time of flash-light stimulation. The pSTR was measured at the position marked by dash line. (B) Mean pSTR and (C) scotopic amplitudes in different experimental groups. Intact group corresponds to undamaged samples. Twelve different biological replicated were used for each group. P value are shown above the graph; ns, not statistically significant. Data are shown as mean ± SEM. One-way ANOVA test was used.

Effects of ATF3 overexpression on the pSTR and scotopic responses 7 dpc. (A) Representative traces of the observable pSTR at different light intensities. *The time of flash-light stimulation. The pSTR was measured at the position marked by dash line. (B) Mean pSTR and (C) scotopic amplitudes in different experimental groups. Intact group corresponds to undamaged samples. Twelve different biological replicated were used for each group. P value are shown above the graph; ns, not statistically significant. Data are shown as mean ± SEM. One-way ANOVA test was used.

Figure 4.

Effects of ATF3 overexpression on the pSTR and scotopic responses 7 dpc. (A) Representative traces of the observable pSTR at different light intensities. *The time of flash-light stimulation. The pSTR was measured at the position marked by dash line. (B) Mean pSTR and (C) scotopic amplitudes in different experimental groups. Intact group corresponds to undamaged samples. Twelve different biological replicated were used for each group. P value are shown above the graph; ns, not statistically significant. Data are shown as mean ± SEM. One-way ANOVA test was used.

Effects of ATF3 overexpression on the pSTR and scotopic responses 7 dpc. (A) Representative traces of the observable pSTR at different light intensities. *The time of flash-light stimulation. The pSTR was measured at the position marked by dash line. (B) Mean pSTR and (C) scotopic amplitudes in different experimental groups. Intact group corresponds to undamaged samples. Twelve different biological replicated were used for each group. P value are shown above the graph; ns, not statistically significant. Data are shown as mean ± SEM. One-way ANOVA test was used.

Figure 5.

Effects of ATF3 overexpression and PTEN deletion on RGC survival and axon regeneration after ONC. (A) Representative images of areas of whole-mount retinas stained with antibodies against RBPMS at 14 dpc. (B) Quantification of RGCs. The number of RGCs/mm2 is shown. (C) Representative images of optic nerve sections at 14 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods; crush sites are indicated with asterisks. (D) Quantification of regenerating axons as in C. Six and four different biological replicates were used for PLAP/PTEN control group and ATF3/PTEN, respectively. Data are shown as mean ± SEM. Unpaired t-test was used for B; ns, not statistically significant. Two-way ANOVA test was used for D. No statistically significant differences were detected at a distance >0.2 mm.

Effects of ATF3 overexpression and PTEN deletion on RGC survival and axon regeneration after ONC. (A) Representative images of areas of whole-mount retinas stained with antibodies against RBPMS at 14 dpc. (B) Quantification of RGCs. The number of RGCs/mm2 is shown. (C) Representative images of optic nerve sections at 14 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods; crush sites are indicated with asterisks. (D) Quantification of regenerating axons as in C. Six and four different biological replicates were used for PLAP/PTEN control group and ATF3/PTEN, respectively. Data are shown as mean ± SEM. Unpaired _t_-test was used for B; ns, not statistically significant. Two-way ANOVA test was used for D. No statistically significant differences were detected at a distance >0.2 mm.

Figure 5.

Effects of ATF3 overexpression and PTEN deletion on RGC survival and axon regeneration after ONC. (A) Representative images of areas of whole-mount retinas stained with antibodies against RBPMS at 14 dpc. (B) Quantification of RGCs. The number of RGCs/mm2 is shown. (C) Representative images of optic nerve sections at 14 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods; crush sites are indicated with asterisks. (D) Quantification of regenerating axons as in C. Six and four different biological replicates were used for PLAP/PTEN control group and ATF3/PTEN, respectively. Data are shown as mean ± SEM. Unpaired _t_-test was used for B; ns, not statistically significant. Two-way ANOVA test was used for D. No statistically significant differences were detected at a distance >0.2 mm.

Effects of ATF3 overexpression and PTEN deletion on RGC survival and axon regeneration after ONC. (A) Representative images of areas of whole-mount retinas stained with antibodies against RBPMS at 14 dpc. (B) Quantification of RGCs. The number of RGCs/mm2 is shown. (C) Representative images of optic nerve sections at 14 dpc. CTB-labeled regenerating axons were visualized as described in Materials and Methods; crush sites are indicated with asterisks. (D) Quantification of regenerating axons as in C. Six and four different biological replicates were used for PLAP/PTEN control group and ATF3/PTEN, respectively. Data are shown as mean ± SEM. Unpaired t-test was used for B; ns, not statistically significant. Two-way ANOVA test was used for D. No statistically significant differences were detected at a distance >0.2 mm.

Figure 6.

mTOR signaling inhibits endogenous expression of ATF3. (A) Transverse sections of control retina (intact) and retina 3 dpc with or without PTEN deletion. Sections were stained with indicated antibodies. (B) Representative images of whole-mount, nonoptic nerve crush retinas immunohistochemically stained for ATF3, RBPMS, and pS6 7 days after intravitreal injections of corresponding rAAV viruses. (C) Percentage of ATF3 positive RGCs in PTEN+/+ and PTEN−/− retinas at 3 dpc. (D) Percentage of ATF3 positive αRGCs in PTEN+/+ and PTEN−/− retinas at 3 dpc. Data are shown as mean ± SEM. Unpaired t-test was used for C and D. N = 3 for each group in A-D.

mTOR signaling inhibits endogenous expression of ATF3. (A) Transverse sections of control retina (intact) and retina 3 dpc with or without PTEN deletion. Sections were stained with indicated antibodies. (B) Representative images of whole-mount, nonoptic nerve crush retinas immunohistochemically stained for ATF3, RBPMS, and pS6 7 days after intravitreal injections of corresponding rAAV viruses. (C) Percentage of ATF3 positive RGCs in PTEN+/+ and _PTEN_−/− retinas at 3 dpc. (D) Percentage of ATF3 positive αRGCs in PTEN+/+ and _PTEN_−/− retinas at 3 dpc. Data are shown as mean ± SEM. Unpaired _t_-test was used for C and D. N = 3 for each group in A-D.

Figure 6.

mTOR signaling inhibits endogenous expression of ATF3. (A) Transverse sections of control retina (intact) and retina 3 dpc with or without PTEN deletion. Sections were stained with indicated antibodies. (B) Representative images of whole-mount, nonoptic nerve crush retinas immunohistochemically stained for ATF3, RBPMS, and pS6 7 days after intravitreal injections of corresponding rAAV viruses. (C) Percentage of ATF3 positive RGCs in PTEN+/+ and _PTEN_−/− retinas at 3 dpc. (D) Percentage of ATF3 positive αRGCs in PTEN+/+ and _PTEN_−/− retinas at 3 dpc. Data are shown as mean ± SEM. Unpaired _t_-test was used for C and D. N = 3 for each group in A-D.

mTOR signaling inhibits endogenous expression of ATF3. (A) Transverse sections of control retina (intact) and retina 3 dpc with or without PTEN deletion. Sections were stained with indicated antibodies. (B) Representative images of whole-mount, nonoptic nerve crush retinas immunohistochemically stained for ATF3, RBPMS, and pS6 7 days after intravitreal injections of corresponding rAAV viruses. (C) Percentage of ATF3 positive RGCs in PTEN+/+ and PTEN−/− retinas at 3 dpc. (D) Percentage of ATF3 positive αRGCs in PTEN+/+ and PTEN−/− retinas at 3 dpc. Data are shown as mean ± SEM. Unpaired t-test was used for C and D. N = 3 for each group in A-D.

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