Retinal structure measured by SD-OCT was altered in the T17M <b><i>RHO</i></b><b> CHOP−/− retina.</b>

<p>We analyzed 4 groups of animals (N = 6) by two-way ANOVA and found significant changes in the average thickness of the ONL in the inferior and superior hemispheres in 1,2 and 3-month-old mice. A: In the superior region, the average thickness of the ONL was 53.89 µm ±0.8 in wild-type, vs. 54.2 µm ±0.2 in CHOP−/− mice. A dramatic reduction of 22% in the ONL thickness was observed between the T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− retinas (29.88 µm ±0.3 in T17M <i>RHO</i> vs. 23.7 µm ±0.3 in T17M <i>RHO</i> CHOP−/−), which was statistically significant (<i>P</i><0.001). The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were also statistically significant (<i>P</i><0.0001). No difference in the thickness of the superior ONL was observed when wild-type and CHOP−/− retinas were compared. At 2 months, the ONL thickness in the T17M <i>RHO</i> retina continued to decline and was 15.83 µm ±0.95 in T17M <i>RHO</i> vs. 12.19 µm ±0.43 in T17M <i>RHO</i> CHOP−/−), which was not statistically significant. In wild- type animals, the ONL thickness was 47.8 µm ±0.38 vs 46.92 µm ±0.37 in CHOP−/− mice. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were also statistically significant (<i>P</i><0.0001). No difference in the thickness of the superior ONL was observed when wild-type and CHOP−/− retinas were compared. At 3 months of age, the ONL thickness in the T17M <i>RHO</i> retina was 17.72 µm ±2.59 in T17M <i>RHO</i> vs. 15.68 µm ±0.43 in T17M <i>RHO</i> CHOP−/−), which was not statistically significant. In wild- type animals, the ONL thickness was 47.15 µm ±0.44 vs 48.47 µm ±0.37 in CHOP−/− mice. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were also statistically significant (<i>P</i><0.0001). No difference in the thickness of the superior ONL was observed when wild-type and CHOP−/− retinas were compared. B: The average thickness of the inferior ONL was also measured in the 4 groups of mice. We found that the average thickness was 52.5 µm ±0.51 in wild-type; 53.4 µm ±0.3 in CHOP−/− mice vs. 31.5 µm ±0.2 in T17M <i>RHO</i>; and 24.5 µm ±0.4 in T17M <i>RHO</i> CHOP−/−. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were statistically significant (<i>P</i><0.0001). The difference (24%) between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− was also statistically significant (<i>P</i><0.001). No difference in the thickness of the inferior ONL was observed when wild-type and CHOP−/− retinas were compared. The average inferior ONL thickness in 2 month-old animals was different in all groups and was 48.0 µm ±0.46 in wild-type; 47.3 µm ±0.75 in CHOP−/− mice vs. 16.7 µm ±0.8 in T17M <i>RHO</i>; and 15.4 µm ±0.4 in T17M <i>RHO</i> CHOP−/−. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were statistically significant (<i>P</i><0.0001). The difference between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− was also statistically significant (<i>P</i><0.001). No difference in the thickness of the inferior ONL was observed when wild-type and CHOP−/− retinas were compared. At 3 months of age the difference in the average inferior ONL thickness was not significant between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− mice (14.4 µm ±0.8 in T17M <i>RHO</i> vs 15.6 µm ±0.4 in T17M <i>RHO</i> CHOP−/−) while differences between wild-type (47.6 µm ±0.8) and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− (48.4 µm ±0.3) and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were statistically significant (<i>P</i><0.0001). No difference in the thickness of the inferior ONL was observed when wild-type and CHOP−/− retinas are compared. C: Histological analyses of wild-type, T17M RHO, T17M RHO CHOP−/− and CHOP−/− retinas: Images of wild-type, T17M RHO, T17M RHO CHOP−/− and CHOP−/− retinas stained with hematoxylin and eosin (H&E). Four animals in each group were used in this experiment. Histology of experimental mouse retinas at 1 month of age showed loss of photoreceptor cell nuclei, shortening of the outer segments, and general disorganization in the T17M RHO retina. Ablation of the CHOP protein in these retinas, however, led to more rapid retinal degeneration that resulted from the shortening of the outer and inner segments and more pronounced general retinal disorganization in the T17M RHO CHOP−/− mice. GCL, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments. Scale bar indicates 50 µm. D: Photoreceptor cell nuclei in all 4 groups of animals. The number of nuclei was counted by a masked researcher. Two-way Anova with multiple comparison analysis demonstrated differences in all 4 groups of animals (****, P<0.0001) at 1 month of age with the exception of comparison between wild-type and CHOP−/− mice. For example, one-month-old T17M RHO mice had 6.7±0.15 rows whereas the T17M RHO CHOP−/− mice had more severe loss of photoreceptor cells with 4.5±0. 21 rows. These numbers were significantly different from those in the wild type and CHOP−/− animals (9.6±0.31 and 10.0±0.41 respectively). Histological analysis confirmed our OCT data suggesting first, that there is a decline in the number of photoreceptor cells in T17M RHO at 1month and second, the CHOP ablation expedites retinal degeneration in T17M RHO retina. Decline in the number of photoreceptors in these animals was 33% compared to T17M RHO mice. E: Immunohistological analyses of one-month-old wild-type, T17M <i>RHO</i>, T17M <i>RHO</i> CHOP−/− and CHOP−/− retinas. 12 micron cryostat sections of retinas were treated with anti-rhodopsin antibody (in green). The IHC analysis revealed normal localization of rhodopsin in the outer segments of photoreceptor cells in wild type and CHOP−/− retinas. The T17M <i>RHO</i> retinas demonstrated shortening of the OS of photoreceptors (propidium iodide -stained ONL nuclei in red). In the T17M RHO <i>CHOP−/−</i> retina we detected mislocalization of rhodopsin (in yellow) in addition to the shortening of the OS of photoreceptors. Rhodopsin was found in the cytoplasm of photoreceptors around the nuclei (in yellow ONL layer) and this, evidently indicates more severe retinal degeneration compared to T17M <i>RHO</i> mice. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments. Scale bar indicates 50 µm.</p

Ablation of C/EBP Homologous Protein Does Not Protect T17M <i>RHO</i> Mice from Retinal Degeneration

<div><p></p><p>Despite the proposed link between ablation of the CHOP protein and delay of the onset of ER stress-mediated disorders including diabetes, Alzheimer Disease, and cardiac hypertrophy, the role of CHOP protein in photoreceptor cell death associated with Autosomal Dominant Retinitis Pigmentosa (ADRP) has not been investigated. T17M <i>RHO</i> transgenic mice carry a mutated human rhodopsin transgene, the expression of which in retina leads to protein misfolding, activation of UPR and progressive retinal degeneration. The purpose of this study is to investigate the role of CHOP protein in T17M <i>RHO</i> retina. Wild-type, CHOP−/−, T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/−mice were used in the study. Evaluation of the impact of CHOP ablation was performed using electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), quantitative Real-Time PCR (qRT-PCR) and western blot analysis. Dark-adapted ERG analysis demonstrated that by 1 month, the T17M <i>RHO</i> CHOP−/− mice had a 70% reduction of the a-wave amplitude compared to the T17M <i>RHO</i> mice. The loss of function in T17M <i>RHO</i> CHOP−/− photoreceptors was associated with a 22–24% decline in the thickness of the outer nuclear layer. These mice had significant reduction in the expression of transcription factors, <i>Crx</i> and <i>Nrl</i>, and also in mouse <i>Rho</i>, and human <i>RHO.</i> The reduction was associated with an 8-fold elevation of the UPR marker, p-eIf2α protein and 30% down-regulation of sXbp1 protein. In addition, the histone deacetylase 1 (Hdac1) protein was 2-fold elevated in the T17M <i>RHO</i> CHOP−/− retina. The ablation of CHOP led to a reduction in the expression of photoreceptor-specific transcriptional factors, and both endogenous and exogenous <i>RHO</i> mRNA. Thus, despite its role in promoting apoptosis, CHOP protects rod photoreceptors carrying an ADRP mutation.</p> </div

CHOP ablation in P30 T17M <b><i>RHO</i></b><b> retinas modulates the expression of Rhodopsin (m-Rho and h-Rho) and photoreceptor-specific transcription factor (Crx and Nrl) genes.</b>

<p>We analyzed 4 groups of animals (N = 6) and found differences in the expression levels of <i>Crx</i>, <i>Nrl</i> and <i>RHO</i> (mRho and T17M <i>RHO</i>). A: mRho and T17M <i>RHO</i> expression was modulated in T17M <i>RHO</i> CHOP−/− retinas. The relative expression of endogenous mouse <i>Rho</i> was 0.9±0.03 in wild-type; 0.9±0.06 in CHOP−/−; 0.4±0.03 in T17M <i>RHO</i>; and 0.02±0.01 in T17M <i>RHO</i> CHOP−/− mice. The differences between wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were statistically significant (<i>P</i><0.001). The 96% reduction of endogenous <i>RHO</i> gene expression, observed in T17M <i>RHO</i> CHOP−/− mice compared to T17M <i>RHO</i> mice was statistically significant (<i>P</i><0.001). No difference in <i>Crx</i> gene expression was observed when the expression of mRho mRNA was compared in wild-type and CHOP retinas. The expression of the human T17M <i>RHO</i> transgene was also modulated in T17M <i>RHO</i> CHOP−/− mice, with a value of 1.1±0.06 in T17M <i>RHO</i> mice vs. 0.04±0.01 in T17M <i>RHO</i> CHOP−/− mice. The observed 96% reduction of transgene expression was statistically significant (<i>P</i><0.0001). B: Modified Expressions of <i>Crx</i> and <i>Nrl</i> in the T17M <i>RHO</i> CHOP−/− retina. The relative <i>Crx</i> gene expression was 1.0±0.059 in wild-type; 1.1±0.1 in CHOP−/−; 0.7±0.03 in T17M <i>RHO;</i> and 0.3±0.02 in T17M <i>RHO</i>. The differences between the wild-type or CHOP−/− and T17M <i>RHO</i> groups were statistically significant (<i>P</i><0.01), as were the differences between wild-type or CHOP−/− and T17M <i>RHO</i> CHOP−/− (<i>P</i><0.001). The 60% reduction of <i>Crx</i> gene expression detected in T17M <i>RHO</i> CHOP−/− retinas compared to T17M <i>RHO</i> was statistically significant (P<0.001). No difference was observed when the wild-type and CHOP−/− retinas were compared. The level of <i>Nrl</i> gene expression was 1.2±0.2 in wild-type; 1.04±0.2 in CHOP−/−; 0.4±0.15 in T17M <i>RHO;</i> and 0.02±0.03 in T17M RHO CHOP−/−. The differences between wild-type and T17M <i>RHO</i> CHOP−/− and CHOP−/− and T17M <i>RHO</i> CHOP−/− were significant at the <i>P</i><0.001 level, while those between wild-type and T17M <i>RHO,</i> T17M <i>RHO</i> and CHOP−/−, and T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− were significant at the <i>P</i><0.01 level. The 95% reduction of <i>Nrl</i> gene expression observed in the T17M <i>RHO</i> CHOP−/− compared to the T17M RHO retina was statistically significant (<i>P</i><0.05). No difference in <i>Nrl</i> gene expression was observed when wild-type and CHOP−/− retinas were compared.</p

Ablation of CHOP protein in P30 T17M <b><i>RHO</i></b><b> retinas led to modulation of the PERK and IRE1 pathways of the UPR.</b>

<p>A: We analyzed T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− retinas (N = 4) and found that the expression of phosphorylated eIF2α protein was increased by over 8 fold in T17M <i>RHO</i> CHOP−/− mice (0.5±0.1 a.u.vs. 0.04±0.1 a.u., <i>P</i> = 0.01). The level of spliced Xbp1 in the T17M <i>RHO</i> retina was decreased by 30%, presenting a value of 0.64±0.027 a.u. vs. 0.4±0.03 a.u.in T17M <i>RHO</i> CHOP−/− mice (<i>P</i> = 0.004). B: Representative images of western blots treated with antibodies against Xbp1, peIF2α and β-actin proteins.</p

Lack of CHOP protein does not protect T17M <b><i>RHO</i></b><b> retinas from degeneration, as measured by scotopic ERG responses at 10DB.</b>

<p>We analyzed 4 groups of animals (N = 6). A: The a-wave of the scotopic ERG amplitude was diminished in T17M <i>RHO</i> CHOP−/− mice at 1 month of age and the values of a-wave amplitudes were 456.3 µv ±39.7 in wild-type; 509.1 µv ±24.9 in CHOP−/−; 169.0 µv ±7.9 in T17M <i>RHO</i>; and 54.4 µv ±16.4 in T17M <i>RHO</i> CHOP−/−. This data reflected a 70% difference in the a-wave amplitudes between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− mice. The differences between all groups were statistically significant (<i>P</i><0.0001). The difference between wild-type and CHOP−/− mice was not significant (n.s.). In the 2^nd and the 3^rd months, the a-wave amplitudes declined in T17M <i>RHO</i> mice but did not decline any further in T17M <i>RHO</i> CHOP−/− mice (90.7 µv ±13.5 in T17M <i>RHO</i> vs. 82.05 µv ±8.7 in T17M <i>RHO</i> CHOP−/− at 2 months and 39.7 µv ±5.7 in T17M <i>RHO</i> vs. 44.0 µv ±8.20 in T17M <i>RHO</i> CHOP−/− at 3 months). In wild-type mice, the a-wave amplitude was 438.2±25.4, vs. 472 µv ±17.3 in CHOP−/− mice at 2 months, while it was 416.2 µv ±45.5 in wild-type vs. 464.9 µv ±35.2 in CHOP−/− mice at 3 months. At 2 and 3 months, the differences between wild-type or CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− groups were significant (<i>P</i><0.001), but we did not register any differences between wild-type and CHOP−/− mice at 1, 2, or 3 months of age and difference between T17M RHO and T17M RHO CHOP−/− mice at 2 and 3 months of age. B: The b-wave of the scotopic ERG amplitude was decreased in T17M <i>RHO</i> CHOP−/− mice over the 3 examined months when compared to T17M <i>RHO</i> retinas. In 1-month-old animals, the b-wave amplitudes were 1023.0 µv ±41.6 in wild-type; 1052.0 µv ±96.5 in CHOP−/−; 475.6 µv ±50.7 in T17M <i>RHO</i>; and 358.2 µv ±58.2 in T17M <i>RHO</i> CHOP−/−. The differences between all groups were statistically significant (<i>P</i><0.0001), except between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/−, which was not significant. In the 2^nd and 3^rd months, the b-wave amplitudes in the T17M <i>RHO</i> mice declined. However, in the T17M <i>RHO</i> CHOP−/− mice, they were consistently low during the next 2 months. The b-wave amplitudes were 407.1 µv ±41.7 in T17M <i>RHO</i> vs. 428.7.5 µv ±67.0 in T17M <i>RHO</i> CHOP−/− at 2 months and 315.3 µv ±32.7 in T17M <i>RHO</i> vs. 214.9 µv ±40.0 in T17M <i>RHO</i> CHOP−/− at 3 months and were not significantly different. No difference was detected between the wild-type and CHOP−/− mice (1028.0 µv ±73.4 in wild-type vs. 894.7 µv ±56.9 in CHOP−/− at 2 months and 982.4 µv ±26.4 in wild-type vs. 916.2 µv ±55.5 in CHOP−/− at 3 months). However, at 2 months, the differences between the wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− mice were significant at the <i>P</i><0.0001 level as were those between the CHOP−/− and T17M <i>RHO</i> mice, while the differences between the CHOP−/− and T17M <i>RHO</i> CHOP−/− mice were significant at the <i>P</i><0.01 level. At 3 months, the differences between the wild-type and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− mice were significant at the <i>P</i><0.00001 level, while those between CHOP−/− and T17M <i>RHO</i> or T17M <i>RHO</i> CHOP−/− were significant at the <i>P</i><0.0001 level. The difference in the b-wave of the ERG amplitude between T17M <i>RHO</i> and T17M <i>RHO</i> CHOP−/− mice was not significant at 3 months. C: Images of the scotopic ERG amplitudes registered at 0 DB or 2.5 cd*s/m² (in red), 10 DB or 25 cd*s/m² (in blue) and 15 DB or 79.1 cd/m² (in green) in four groups of animals.</p

Expressions of the histone deacetylase, Hdac1 and the transcriptional co-activator, P300 were modified in P30 T17M <b><i>RHO</i></b><b> CHOP−/− retinas (N = 3).</b>

<p>A: A 78% reduction in the P300 protein level was observed in the T17M <i>RHO</i> CHOP−/− retina compared to T17M <i>RHO</i>. The normalized level of P300 was 0.3±0.06 a.u. in T17M <i>RHO</i> vs. 0.1±0.02 a.u. in T17M <i>RHO</i> CHOP−/− (P = 0.02). B: A 245% increase in Hdac1 protein expression was observed in T17M <i>RHO</i> CHOP−/− retinas (0.22±0.05 arbitrary units in T17M <i>RHO</i> vs. 0.54±0.08 a. u. in T17M <i>RHO</i> CHOP−/−, <i>P</i> = 0.03). Bottom: Representative images of western blots treated with antibodies against P300, HDAC1 and β-actin proteins.</p

Proposed mechanisms of retinal degeneration in T17M and T17M ATF4^+/- mice.

<p>In T17M retinas, we observed up-regulation of pPERK, ATF4, CHOP, p53 proteins resulting in activation of apoptosis. In T17M retinas deficient in ATF4 we detected up-regulation of peIF4E, NRF2, GADD34 and autophagy genes resulting in delay of retinal degeneration. Increased and decreased levels of proteins in the retina are present as small and large fonts, correspondingly. Increased and decreased effects in solid and dashed lines, respectively are shown as well. Proposed in the literature links between signaling molecules are showed by numbers corresponding to their citations in the References section.</p

ATF4 deficiency in P30 T17M retinas results in upregulation of autophagosome associated genes.

<p><b>(A)</b> Level of autophagy–related LC3-II (lipidated form) was diminished in T17M retinas as compared to C57BL6, ATF4^+/- and T17M ATF4^+/- retinas (<i>P</i> = 0.005, <i>P</i><0.01 and <i>P</i><0.05 respectively). Beclin-1 protein significantly up-regulated in T17M ATF4^+/- retinas as compared to C57BL6 and T17M retinas (<i>P</i><0.05 and <i>P</i><0.01 respectively). The T17M ATF4^+/-retinas showed increased peIF4E protein levels as compared to T17M retinas (P<0.05). The data are presented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a> for details.</p

Overexpression of ATF4 accelerates and induces retinal degeneration in T17M mice and C57BL6 mice, respectively.

<p><b>(A</b>) ERG amplitudes were registered as described in the methods and were analyzed by one-way ANOVA. Data analysis demonstrated that subretinal injections of AAV2/5- ATF4 led to a loss of photoreceptor function in the right ATF4-injected T17M retina compared to the left AAV2/5- GFP-injected eye (N = 6, <i>P</i><0.05 for both a and b-wave amplitudes). The C57BL6 retina overexpressing ATF4 also experienced a decline in a- and b- wave ERG amplitudes (N = 4, for both <i>P</i><0.01) as compared to GFP-injected controls or uninjected animals (N = 4). Results of the scotopic ERG amplitudes registered at 25 cd*s/m2 are shown. <b>(B)</b> Overexpression of ATF4 in the injected (right) retinas was measured by western blotting. A 2.3-fold (N = 6, <i>t</i>-test p = 0.017) increase in ATF4 was found in the right eye when compared to the left eye. This resulted in upregulation of CHOP protein (N = 6, <i>t</i>-test p = 0.046) in ATF4 overexpressing eyes. Representative images of western blots probed with antibody against ATF4, CHOP, and β-actin are shown on the side. <b>(C)</b> Overexpression of ATF4 and CHOP proteins in T17M and C57BL6 retinas was associated with a loss of photoreceptor cells measured by counting the nuclei rows in H&E stained retinal sections (N = 4, <i>t</i>-test <i>P</i><0.001 and N = 3, <i>t</i>-test <i>P</i><0.001). <b>(D)</b> Representative images of the H&E stained T17M and C57BL6 retinas injected with AAV2/5-GFP and AAV2/5-ATF4. <b>(E)</b> Photoreceptor cell death in T17M retinas overexpressing ATF4 was associated with highly activated caspase-3/7. The activation of apoptotic cell death markers was also detected in the wild type retinas over-expressing ATF4 (N = 4, <i>t-</i>test <i>P</i><0.001 and <i>P</i><0.0001, respectively). The data are presented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a>.</p

ATF4 knockdown protects T17M mice from loss of retinal integrity and photoreceptors.

<p>(<b>A)</b> We found dramatic increases in the average ONL thicknesses of both hemispheres of 1, 2, and 3-month-old T17M ATF4^+/- retinas compared to T17M mice (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154779#pone.0154779.s004" target="_blank">S1 Table</a>). Representative spidergrams of the distribution of ONL thicknesses across the retina at 1, 2, and 3 months of age are shown. The data were analyzed by two-way ANOVA. The T17M ATF4^+/- retinas (N = 8) demonstrated an increase in the superior and inferior ONL thickness at 1, 2, and 3 months of age as compared to T17M mice (N = 11). All regions from both ADRP retinas were significantly different from C57BL6 (N = 7) and ATF4 (N = 7) mice (<i>P</i><0.0001 for both strains and all regions). The data are presented as mean ± SEM. (<b>B</b>) The ATF4 deficiency in T17M mice protected their retinas from photoreceptor cell loss, resulting in an increase in the number of photoreceptors relative to T17M as measured by two way ANOVA (<i>P</i><0.0001). The number of rows of photoreceptor nuclei between T17M ATF4^+/- (N = 4) and C57BL6 (N = 4) retinas was similar at P30, whereas the T17M mice lost 43% of their photoreceptor cells (<i>P</i><0.0001 as compared to all groups). However, by 3 months, both the T17M ATF4^+/- and the T17M mice experienced a loss of photoreceptor cells as compared to C57BL6 mice (<i>P</i><0.0001). The data are presented as mean ± SEM. <b>(C)</b> Representative images of H&E stained retinal sections from all four groups. Images of one-month-old (upper) and 3-month-old (bottom) control and experimental retinas. Scale bar indicates 50 μm.</p