CRALBP supports the mammalian retinal visual cycle and cone vision

Abstract

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.

Figure 8. Deletion of CRALBP reduces the threshold of the pupillary light reflex.

(A) Comparison of pupil size in darkness and in the light (~14 log photons/cm²/s) in control, Rlbp1^–/–, and Rpe65^–/– (Gnat1^+/+) negative control mice. (B) Averaged intensity-response curves for control (n = 5), Rlbp1^–/– (n = 5), and Rpe65^–/– (n = 4) mice. A significant difference was observed at threshold (*P < 0.05) between Rlbp1^–/– and control mice by 2-way ANOVA followed by Bonferroni’s post test. Results represent the mean ± SEM. (C) Intensity required to reach EC₅₀ in control and Rlbp1^–/– mice. P = 0.10, NS, by 2-tailed unpaired Student’s t test.

Figure 7. AAV-driven expression of CRALBP in Müller cells rescues the sensitivity and dark adaptation of CRALBP-deficient cones.

(A) Ensemble-averaged transretinal cone intensity-response curves for Rlbp1^–/– mice injected with AAV driving expression of GFP in RPE or Müller cells (black squares, n = 4), CRALBP in Müller cells (red circles, n = 5), and CRALBP in RPE cells (green diamonds, n = 3). (B) Transretinal dim flash responses of Rlbp1^–/– mice showing the relative amplification for AAV-driven control GFP (black, n = 4), Müller cell–specific CRALBP (red, n = 5), and RPE-specific CRALBP (green, n = 3) expression. (C) In vivo ERG recordings of cone b-wave dark adaptation (b-wave S_f/b-wave S_f^DA) after a 90% bleaching in Rlbp1^–/– mice with AAV-driven expression of control GFP (black, n = 11), Müller cell–specific CRALBP (red, n = 12), and RPE-specific CRALBP (green, n = 8). (D) Transretinal recordings of cone dark adaptation (S_f/S_f^DA) after a 90% bleaching of Rlbp1^–/– retinae with AAV-driven expression of control GFP (black, n = 4), Müller cell–specific CRALBP (red, n = 5), and RPE-specific CRALBP (green, n = 3). Results represent the mean ± SEM.

Figure 6. AAV-mediated delivery of CRALBP to Müller cells or RPE cells.

Antibody staining shows expression pattern of CRALBP in (A) control retina, (B) Rlbp1^–/– retina, (C) Müller cells of Rlbp1^–/– retina after intravitreal injection with an AAV construct targeted for Müller cells shH10-scCAG-RLBP1, and (D) RPE of Rlbp1^–/– retina after intravitreal injection with an AAV construct targeted for RPE 7m8-scVMD2-RLBP1. Scale bars: 50 μm. INL, inner nuclear layer; GCL, ganglion cell layer. Widespread infection across the retina was achieved for both constructs, as seen in tiled images for (E) shH10-scCAG-RLBP1 and (F) 7m8-scVMD2-RLBP1. Red channel, anti-CRALBP; blue channel, DAPI. Scale bar: 200 μm.

Figure 5. Deletion of CRALBP affects the localization of M-opsin and number of cones expressing M-opsin.

Antibody staining of retinal frozen sections from Rlbp1^–/– and control mice for (A) M-opsin and (B) S-opsin. Representative images are shown. At least 3 retinae per condition were examined. Scale bars: 25 μm. COS, cone outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer. For clarity, the DAPI channel is not shown. (C) Quantification of whole-mount M-opsin antibody staining (n = 4 retinae per condition). *P < 0.05, **P < 0.01 by unpaired 2-tailed Student’s t test. (D) Quantification of whole-mount S-opsin antibody staining (n = 3 retinae per condition). D, dorsal; T, temporal; N, nasal; V, ventral. Young, 6- to 7-week-old mice; Old, 3- to 6-month-old mice. Results represent the mean ± SEM.

Figure 4. Dark rearing, but not acute treatment with exogenous chromophore, rescues CRALBP-deficient cone sensitivity.

(A) Normalized in vivo ERG cone b-wave intensity-response curves for untreated control (replotted from Figure 2B inset) and 9-cis retinal–treated (n = 6) Rlbp1^–/– mice. (B) Normalized transretinal cone intensity-response curves for control (black, n = 6) and Rlbp1^–/– (red, n = 6) retinae in control solution (filled symbols; replotted from Figure 3B inset) and after treatment with exogenous 11-cis retinal (open symbols, n = 6). 9cRal, 9-cis retinal; 11cRal, 11-cis retinal. (C) Cone b-wave intensity-response curves from in vivo ERG recordings of control mice raised in cyclic light (black squares, n = 14) or in darkness (white squares, n = 10). (D) Cone b-wave intensity-response curves from in vivo ERG recordings of control (black squares; replotted from Figure 1B) and Rlbp1^–/– mice raised in cyclic light (red filled circles; replotted from Figure 1B) and Rlbp1^–/– mice raised in darkness (open red circles, n = 10). Insets in C and D show the corresponding normalized intensity-response curves. (E) Normalized cone b-wave sensitivity (b-wave S_f / b-wave S_f^DA) from in vivo ERG recordings during dark adaptation following 90% pigment bleaching at t = 0 for control (black squares) and Rlbp1^–/– mice raised in cyclic light (filled red circles; replotted from Figure 4A) and for Rlbp1^–/– mice raised in darkness (open red circles, n = 10). Results represent the mean ± SEM.

Figure 3. The deletion of CRALBP suppresses cone dark adaptation.

(A) Normalized cone b-wave sensitivity (b-wave S_f /b-wave S_f^DA) from in vivo ERG recordings during dark adaptation following 90% pigment bleaching at t = 0 for control (n = 10) and Rlbp1^–/– (n = 10) mice. (B) Cone sensitivity, S_f, normalized to its dark-adapted value, S_f^DA, from transretinal recordings during dark adaptation following 90% pigment bleaching at t = 0 for control (n = 9) and Rlbp1^–/– (n = 10) isolated retinae. Results represent the mean ± SEM.

Figure 2. Deletion of CRALBP reduces transretinal cone-response amplitude and sensitivity.

(A) Representative transretinal cone responses from control (left panel) and Rlbp1^–/– (right panel) retinae. Test flash intensities increased from 23 photons/μm² to 1.40 × 10⁶ photons/μm² in steps of 0.5 log units. For both panels, the flash intensity producing the response shown in red was 1.39 × 10⁴ photons/μm². (B) Ensemble-averaged absolute and normalized (inset) cone intensity-response curves for control (n = 13) and Rlbp1^–/– (n = 13) retinae. (C) Ensemble-averaged normalized cone dim flash responses from control (n = 12) and Rlbp1^–/– (n = 13) retinae. (D) Ensemble-averaged dim flash responses, r, from control (n = 13) and Rlbp1^–/– (n = 11) cones normalized to the maximal response, r_max, and to flash intensity and with matched rising slopes to determine the change in phototransduction amplification. Results are shown as the mean ± SEM.

Figure 1. Deletion of CRALBP reduces photopic in vivo ERG–response amplitude and sensitivity.

(A) Representative in vivo cone ERG responses from control (black traces), Rlbp1^–/– (red traces), and Rlbp1^+/– (blue traces) mice. Test flash intensities increased from 2.27 × 10^–2 cd × s/m² (bottom traces) to 697 cd × s/m² (top traces) in steps of approximately 0.5 log units. (B) Ensemble-averaged cone b-wave intensity-response curves for control (n = 10), Rlbp1^–/– (n = 12), and Rlbp1^+/– (n = 10) mice. (C) Cone b-wave intensity-response curves for control mice aged 6–7 weeks (black squares, n = 8) and 13–16 weeks (white squares, n = 10). (D) Cone b-wave intensity-response curves for Rlbp1^–/– mice aged 6–7 weeks (red circles, n = 6) and 13–16 weeks (white circles, n = 6). Insets in B–D show the corresponding normalized (r/r_max) intensity-response curves. Results represent the mean ± SEM.