RDH12 allows cone photoreceptors to regenerate opsin visual pigments from a chromophore precursor to escape competition with rods

Abstract

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Keywords: RDH12; cone photoreceptor; dehydrogenase; retinal; vision; visual cycle; zebrafish.

Figure 1.. ZCRDH is expressed in zebrafish cone inner segments.

An antibody was generated against an N-terminal peptide ZCRDH (product of the zgc:153441 gene) (Figure S2). (A) The antibody was tested with four different protein homogenates: pcDNA3.1 HEK, HEK-293T cells transfected with the non-recombinant expression plasmid, pcDNA3.1; ZCRDH HEK, HEK-293T cells transfected with plasmid containing the ZCRDH coding region; wild-type ZF eyes: whole eye tissues from wild-type (AB strain) zebrafish; Zcrdh-mutant eyes, whole-eye tissues from Zcrdh-mutant zebrafish. The antibody detected a protein band of the predicted size (~37kD) in retinal homogenates from wild-type and ZCRDH-expressing HEK-293T cells. The slightly higher molecular mass of ZCRDH in HEK-cell versus wild-type zebrafish retina homogenates is due to the presence of a Flag epitope-tag at the C-terminus of recombinant ZCRDH. The same blot was re-probed with an antibody against β-tubulin as a protein loading control, shown below the ZCRDH immunoblot. A similar blot was probed with the ZCRDH antibody after preincubation with the immunizing peptide. Note the absence of signal in this control for Ab specificity. (B) Immunohistochemistry performed on adult wild-type zebrafish retinal sections using ZCRDH Ab (red), peanut agglutinin (PNA) lectin to label the sheath surrounding cone outer segments (green), and the nuclear stain 4′,6-diamidino-2-phenylindole DAPI (blue). ZCRDH immunoreactivity is strongly present in cone inner segments. (C) Immunocytochemistry performed on adult Zcrdh-mutant retinal sections using the same ZCRDH-Ab, PNA and DAPI labels as in (B). The region labeled, ‘rod and cone IS and OS’ corresponds to the region of rod and cone photoreceptor outer and inner segments. The region labeled, ‘ONL’ corresponds to the outer nuclear layer containing rod and cone photoreceptor nuclei. Note the greatly reduced labeling of cone inner segments in Zcrdh-mutant versus wild-type zebrafish retinas. See also Figure S2.

Figure 2.. Histological analysis of retina sections from wild-type and Zcrdh-mutant zebrafish.

Histologic sections from six-months-old wild-type (A) and Zcrdh-mutant (B) zebrafish retinas. Sections were stained with 1% toluidine blue and 1% sodium borate and photographed with a 20x objective. Retina layers are identified to the right of panel (B). RPE: retinal pigment epithelium; rod and cone OS, IS and ONL: rod and cone outer segments, inner segments and outer nuclear layer (containing rod and cone photoreceptor nuclei); OPL: outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; and GCL: ganglion cell layer. Note the similar number of photoreceptor nuclei and similar thickness of retinal layers in wild-type and Zcrdh-mutant retinas.

Figure 3.. Retinoid profiles of Zcrdh-mutant and wild-type zebrafish eyes under different light-exposure conditions.

Whole-eye retinoid profiles of age-matched Zcrdh-mutant (red) and AB wild-type (blue) adult zebrafish (five-month-old) under different light-exposure conditions: DA, overnight dark-adapted; bleached, immediately following an ~50% photobleach; bleach + 5-, 30- or 60-min DA, bleached followed by the indicated period of recovery in the dark. (A) 11cRAL chromophore levels in the eyes of wild-type or Zcrdh-mutant zebrafish after indicated light-exposures. (B) 11cROL levels in the eyes of wild-type or Zcrdh-mutant zebrafish after indicated light-exposures. (C) 11cRP levels in the eyes of wild-type or Zcrdh-mutant zebrafish after indicated light-exposures. Error bars show standard deviation of the mean for four replicates (n = 4).

Figure 4.. Absent regeneration of cone visual pigments in Zcrdh-mutant retinal explants following a photobleach and treatment with 11cROL chromophore-precursor.

Absorbance spectra of wild-type and Zcrdh-mutant zebrafish photoreceptors. (A) Average absorbance spectra from wild-type zebrafish M-cones. The mean peak optical densities (± s.e.m.) were OD_DA = 0.024 ± 0.0025, OD_bleach = 0.0036 ± 0.0033, OD_11cROL = 0.018 ± 0.0043. (B) Average absorbance spectra from wild-type zebrafish L-cones. The mean peak optical densities were OD_DA = 0.030 ± 0.0043, OD_bleach = 0.0091 ± 0.0041, OD_11cROL = 0.021 ± 0.0039. (C) Average absorbance spectra from wild-type zebrafish rods. The mean peak optical densities were OD_DA = 0.044 ± 0.0050, OD_bleach = 0.0076 ± 0.0031, OD_11cROL = 0.0060 ± 0.0019. (D) Average absorbance spectra from Zcrdh-mutant zebrafish M-cones. The mean peak optical densities were OD_DA = 0.021 ± 0.0040, OD_bleach = 0.0023 ± 0.0099, OD_11cROL = 0.0059 ± 0.0038 , OD_11cRAL = 0.017 ± 0.0028. (E) Average absorbance spectra from Zcrdh-mutant zebrafish L-cones. The mean peak optical densities were OD_DA = 0.018 ± 0.0037, OD_bleach = 0.0033 ± 0.0070, OD_11cROL = 0.0052 ± 0.0035, OD_11cRAL = 0.021 ± 0.0017. (F) Average absorbance spectra from Zcrdh-mutant zebrafish rods. The mean peak optical densities were OD_DA = 0.029 ± 0.0028, OD_bleach = 0.0020 ± 0.0028, OD_11cROL = 0.0050 ± 0.0024, OD_11cRAL = 0.023 ± 0.0020. Data from dark-adapted and regenerated cells are fitted with A1 nomograms with the following parameters: WT DA M-cone: λ_max = 492 nm, OD_peak = 0.025; WT 11cROL M-cone: λ_max = 500 nm, OD_peak = 0.017; WT DA L-cone: λ_max = 555 nm, OD_peak = 0.033; WT 11cROL L-cone: λ_max = 547 nm, OD_peak = 0.024; WT DA rod: λ_max = 498 nm, OD_peak = 0.047; Zcrdh-mutant DA M-cone: λ_max = 502 nm, OD_peak = 0.018; Zcrdh-mutant 11cRAL M-cone: λ_max = 491 nm, OD_peak = 0.021; Zcrdh-mutant DA L-cone: λ_max = 554 nm, OD_peak = 0.022; Zcrdh-mutant 11cRAL L-cone: λ_max = 551 nm, OD_peak = 0.023; Zcrdh-mutant DA rod: λ_max = 507 nm, OD_peak = 0.027; Zcrdh-mutant 11cRAL rod: λ_max = 510 nm, OD_peak = 0.022.

Figure 5.. 11cROL-oxidase activity of ZCRDH and various mammalian RDH candidates for the cone 11cROL-oxidase and substrate kinetics for ZCRDH and RDH12.

HEK-293T cells were transfected with non-recombinant pcDNA3.1 or the same plasmid containing the coding regions for the indicated proteins. All clones were made with a C-terminal FLAG-tag. Assays were carried out using homogenate of the transfected cells as an enzyme source with 2 µM 11cROL and 1 mM NADP+ cofactor for one min at 37ºC with gentle agitation. (A) 11cROL-oxidase activities (11cRAL synthesis): zf-ZCRDH, zebrafish ZCRDH; hum-RDH11, human RDH11; hum RDH12, human RDH12; hum-RDH13, human RDH13; and mus-RDH14, mouse RDH14. (B) 11cROL-oxidase activities: zf-ZCRDH-L, zebrafish ZCRDH-like; zf-RDH12, predicted zebrafish RDH12; zf-RDH12-L, predicted zebrafish RDH12-like; carp-ZCRDH, carp ZCRDH; carp RDH13-L, carp RDH13-like. (C) Immunoblots containing equal protein amounts of HEK-293T cell-homogenates transfected with the indicated plasmids and reacted with an anti-FLAG antibody. Note the similar intensities of immunoreactive bands containing the different proteins. The same blot was re-probed with an antibody against β-tubulin as a protein loading control, shown below the anti-FLAG immunoblot. (D) Michaelis-Menton analysis of 11cROL oxidation of ZCRDH and (E) human RDH12 using the indicated 11cROL concentrations and 1 mM NADP+. The assay conditions for kinetic analysis are as described in Figure 5. Calculated K_M and V_max values are shown. Error bars show standard deviation of the mean for three replicates (n = 3). See also Figures S1 and S3, and and S2.

Figure 6.. Comparison of redox-coupled catalytic activities of ZCRDH and human RDH12.

(A) 11cRAL synthesis by ZCRDH and human RDH12 in enzyme assays containing 5 µM of the indicated substrate(s) and no added dinucleotide cofactor. (B) atROL synthesis by ZCRDH and human RDH12 containing 5 µM of the indicated substrate(s) and no added NADP(H) cofactor. Error bars at each concentration show standard deviation of the mean for three replicates (n = 3). Data sets were analyzed by one-way ANOVA followed by Student-Newman-Keuls t-test to test for significance between different test groups. P-value significance is indicated by asterisks. ** = P-value ≤ 0.05; ** = P-value ≤ 0.01; *** = P-value ≤ 0.001. Test group comparisons without indicated P-values are not significant (P-value > 0.05).

Figure 7.. Proposed model for the photic cone visual-cycle in Müller glial cells and cone photoreceptors.

Absorption of a photon (hv) by a cone-opsin pigment isomerizes the 11cRAL to atRAL, activating visual transduction. Shortly afterwards, atRAL dissociates from the opsin, is reduced to atROL by RDH12 and is released into the extracellular space for recycling. The atROL is taken up by the Müller cell where it is re-oxidized by RDH10 to atRAL, which binds covalently to apo-RGR opsin. Absorption of a photon by RGR opsin isomerizes atRAL to 11cRAL. The 11cRAL is reduced, again by RDH10, to 11cROL, which is released by Müller cells into the extracellular space. Finally, the 11cROL is taken up by the cone photoreceptor where it is oxidized by RDH12 to 11cRAL, which combines with cone apo-opsin to form a new cone visual pigment. Since only cones can utilize 11cROL to regenerate their visual pigments, the visual cycle in Müller cells helps cones avoid competition with rods for chromophore under bright-light conditions.