Absence of Sigma 1 Receptor Accelerates Photoreceptor Cell Death in a Murine Model of Retinitis Pigmentosa

Abstract

Purpose: Sigma 1 Receptor (Sig1R) is a novel therapeutic target in neurodegenerative diseases, including retinal disease. Sig1R-/- mice have late-onset retinal degeneration with ganglion cell loss that worsens under stress. Whether Sig1R plays a role in maintaining other retinal neurons is unknown, but was investigated here using rd10 mice, a model of severe photoreceptor degeneration.

Methods: Wild-type, rd10, and rd10/Sig1R-/- mice were subjected to ERG and spectral-domain optical coherence tomography (SD-OCT) to assess visual function/structure in situ. Retinas imaged microscopically were subjected to morphometric analysis, immunodetection of cones, and analysis of gliosis. Oxidative and endoplasmic reticulum (ER) stress was evaluated at mRNA/protein levels.

Results: Photopic ERG responses were reduced significantly in rd10/Sig1R-/- versus rd10 mice at P28 (31 ± 6 vs. 56 ± 7 μV), indicating accelerated cone loss when Sig1R was absent. At P28, SD-OCT revealed reduced retinal thickness in rd10/Sig1R-/- mice (60% of WT) versus rd10 (80% of WT). Morphometric analysis disclosed profound photoreceptor nuclei loss in rd10/Sig1R-/- versus rd10 mice. rd10/Sig1R-/- mice had 35% and 60% fewer photoreceptors, respectively, at P28 and P35, than rd10. Peanut agglutinin cone labeling decreased significantly; gliosis increased significantly in rd10/Sig1R-/- versus rd10 mice. At P21, NRF2 levels increased in rd10/Sig1R-/- mice versus rd10 and downstream antioxidants increased indicating oxidative stress. At P28, ER stress genes/proteins, especially XBP1, a potent transcriptional activator of the unfolded protein response and CHOP, a proapoptotic transcription factor, increased significantly in rd10/Sig1R-/- mice versus rd10.

Conclusions: Photoreceptor cell degeneration accelerates and cone function diminishes much earlier in rd10/Sig1R-/- than rd10 mice emphasizing the importance of Sig1R as a modulator of retinal cell survival.

Figure 1

Scotopic ERG. (A) Scotopic ERG traces. Averaged scotopic ERG responses to 5-ms flashes at a series of intensities in WT, rd10, and rd10/Sig1R^−/− mice at P28; intensities are units of lumens. (B) Mean b-wave amplitudes. Data are the mean ± SEM of assessments of six to nine mice. (C) Enlargement of b-wave amplitude in rd10 and rd10/Sig1R^−/−. *Significant difference between rd10 and rd10/Sig1R^−/− mice (P < 0.05).

Figure 2

Photopic ERG and natural luminance noise test. (A) Photopic ERG traces. Averaged photopic responses to 5-ms flashes at a series of contrasts are provided for WT, rd10, and rd10/Sig1R^−/− mice at P28. (B) Mean b-wave amplitudes of averaged photopic responses to 5-ms flashes above a fixed pedestal luminance of 0.105 lumens (four contrasts of the flash; contrast = [flash-pedestal]/pedestal luminance). Data are the mean ± SEM of four assays using eyes from six to nine mice. *Significantly different from the WT and rd10 groups, *P < 0.05. (C) Averaged responses to photopic flash of contrast =1 (replotted after superimposition). (D) Averaged responses to 0.5-second-long luminance steps are shown. The photopic negative response occurs after stimulus offset at 0.5 second, and originates in part from RGCs. (E) Averaged kernels derived from responses to natural noise stimuli. Green, WT mice; red, rd10 mice; blue, rd10/Sig1R^−/− mice.

Figure 3

SD-OCT assessment. (A–D) Representative SD-OCT data obtained from WT mice, rd10 mice, and rd10/Sig1R^−/− mice at P21 (A), P28 (B), P35 (C), and P42 (D). (E–T) Data for segmentation analysis for TRT at P21, P28, P35, and P42 (E–H), for ONL thickness at P21 to P42 (I–L), for IS/OS thickness at P21 to P42 (M–P), and for OPL thickness at P21 to P42 (Q–T). (U) The template (5 × 5 grid) for the OCT measurements. It represents the 25 points within the retina where retinal thickness is measured. Data are the mean ± SEM of analyses in 4 to 10 mice per group at each age. **P < 0.01, ***P < 0.001. Note: For the OCT analyses, the x-axis represents the 25 points shown in (U), with point #13 representing the optic nerve, where the thickness is always zero.

Figure 4

Retinal structure and morphometric analysis. Retinal sections of eyes embedded in JB4 and stained with H&E from WT, rd10, and rd10/Sig1R^−/− mice. (A–G) Representative image from WT, rd10, rd10/Sig1R^−/− groups at P15 (A), P18 (B), P21 (C), P24 (D), P28 (E), P35 (F) and P42 (G). Note accelerated retinal detachment and paucity of PRC in the ONL in rd10/Sig1R^−/− compared with rd10 mice. (H–Y) Morphometric analyses of TRT (H–M), ONL thickness (N–S), and OPL thickness (T–Y) at P18, P21, P24, P28, P35, and P42 separately. gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; is, inner segment; os, outer segment; rpe, retinal pigment epithelium. Data are the mean ± SEM of measurements from six to nine mice per group. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar: 50 μm. Numbers of mice used in the analysis are provided in Supplementary Table S1.

Figure 4

Continued.

Figure 5

Immunodetection of cone PRCs. (A–C) Retinal cryosections subjected to PNA immunolabeling from retinas of WT (A), rd10 (B), and rd10/Sig1R^−/− (C) mice at P35. (D) Quantification of PNA fluorescence. (E–G) Representative PNA-immunolabeled retinal flatmounts from WT (E), rd10 (F), and rd10/Sig1R^−/− (G) mice. (H) Quantification of PNA-positive cells. Data are the mean ± SEM of four assays from six to eight mice (cryosections) and from 7 to 10 mice (flatmounts) (Supplementary Table S1). *P < 0.05; **P < 0.01. Scale bars: 100 μm in (A–C); 50 μm in (E–G). Nuclei are labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Details about numbers of mice used in the analysis are provided in Supplementary Table S1, and details about antibodies are provided in Supplementary Table S2.

Figure 6

Assessment of glial cell activation. (A–C) Immunodetection of GFAP (green) in retinal cryosections from WT (A), rd10 (B), and rd10/Sig1R^−/− mice (C) at P35. Nuclei are labeled with DAPI (blue). (D) Quantification of fluorescence intensity in WT, rd10, and rd10/Sig1R^−/− mice. Data are the mean ± SEM (three or four assays) from six to eight mice per group. *P < 0.05; ***P < 0.001 (Supplementary Table S1). (E–G) Retinal flatmounts immunolabeled with Iba-1 from WT (E), rd10 (F), and rd10/Sig1R^−/− (G) mice. (H) Quantification of Iba-1 positive cells in retinal flatmounts. Data are the mean ± SEM (three or four assays) from six to eight mice per group. *P < 0.05; ***P < 0.001 (Supplementary Table S1). Calibration bars: (A–C): 100 μm, (E–G): 50 μm. Details about antibodies are provided in Supplementary Table S2.

Figure 7

Assessment of oxidative stress–related genes and proteins. Immunodetection of CM-H₂DCFDA (green fluorescence on reaction with reactive oxygen species) in retinal cryosections of (A) WT, rd10, and rd10/Sig1R^−/− at P21. Nuclei are labeled with DAPI (blue). Calibration bar: 50 μm. RNA was isolated from neural retinas and subjected to quantitative RT-PCR (qRT-PCR) analysis of (B) Nrf2, Keap1. Neural retinas harvested from WT, rd10, and rd10/Sig1R^−/− mice at P21 were used for isolation of protein. Representative immunoblots detecting (C) NRF2 and KEAP1. (D) Band densities of NRF2 and KEAP1 quantified densitometrically and expressed as fold change versus glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RNA was isolated from neural retinas and subjected to qRT-PCR analysis of (E) Sod1, Catalase, Nqo1, Gpx1, Hmox1, and Gstt3. (F) Neural retinas harvested from WT, rd10, and rd10/Sig1R^−/− mice at P21 were used for isolation of protein. Representative immunoblots detecting SOD1, Catalase, NQO1, HMOX1. (G) Band densities of SOD1, Catalase, NQO1, HMOX1 quantified densitometrically and expressed as fold change versus β-Actin. Primer pairs for PCR studies are provided in Supplementary Table S3, and antibodies for immunodetection are provided in Supplementary Table S2. Data are the mean ± SEM of three assays from three different mice retinas in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8

Assessment of ER stress–related genes and proteins. (A) RNA was isolated from neural retinas and subjected to quantitative real-time RT-PCR analysis of Ire1α, Xbp1, Atf4, Chop, Bip, Perk, Ip3r3, and Atf6. Primer pairs are listed in Supplementary Table S3. Data are the mean ± SEM of three assays from three different mice retinas in each group. *P < 0.05; **P < 0.01; ***P < 0.001. (B, C) Neural retinas harvested from WT, rd10, and rd10/Sig1R^−/− mice at P21 were used for isolation of protein. Representative immunoblots detecting IRE1α, XBP1, ATF4, CHOP, BiP, PERK, IP3R3, and p-eIF2α/eIF2α are shown in (B) and (C). GAPDH and β-Actin were the internal controls, separately. (D) Band densities, quantified densitometrically and expressed as fold change versus GAPDH or β-Actin. Data are the mean ± SEM of three or four assays from different retina of WT, rd10, and rd10/Sig1R^−/− mice. *P < 0.05; **P < 0.01; ***P < 0.001.