Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 277 (21), 19173-82

Recovery of Visual Functions in a Mouse Model of Leber Congenital Amaurosis


Recovery of Visual Functions in a Mouse Model of Leber Congenital Amaurosis

J Preston Van Hooser et al. J Biol Chem.


The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.


F<sc>ig</sc>. 1
Fig. 1
Changes in retinoid levels and interface between RPE and ROS in Rpe65/mice gavaged with 9-cis-retinal. A, the levels of all-trans-retinyl esters (closed circles) and 11-cis-retinal (closed squares) in Rpe65+/+ compared with levels of all-trans-retinyl esters (open circles) in Rpe65−/− mice as a function of age. B, ester analysis of 9-cis-retinal-treated and untreated Rpe65−/− mice. Rpe65−/− mice were treated with 25 μg of 9-cis-retinal starting at PND 7 every other day until they were 1 month old. Note the y axis scale. C, age-related accumulation of all-trans-retinyl esters in Rpe65−/− mice (gray line with black data points) compared with the ester levels (circles) in animals treated with 9-cis-retinal starting at PND 7 (left panel) (25 μg every other day, and after PND 30 gavaged with 9-cis-retinal (250 μg) once a week) or PND 30 (right panel) gavaged with 9-cis-retinal (250 μg) once a week. The levels of iso-Rh in treated Rpe65−/− mice are indicated by triangles measured as 11-cis-retinyl oximes. D, changes in the RPE-ROS interface in Rpe65 mice treated with 9-cis-retinal. Rpe65−/− mice were treated with 9-cis-retinal (200 μg each) at PND 7, 11, and 15 and analyzed when they were PND 30 (panels c and d) and PND 90 (panels e and f). Rpe65−/− mice were treated with 9-cis-retinal (200 μg each) at PND 30 and analyzed when they were PND 120 (panels g and h). Control retina from untreated Rpe65−/− mice at PND 7 and PND 30 is shown on the top (panels a and b, respectively). Only partially filled lipid-like droplet in early treated mice (left column, red arrow), and considerably improved RPE-ROS processes (right column) in all treated mice were observed. Scale bar, 1 μm.
F<sc>ig</sc>. 2
Fig. 2
Effects of light exposure on iso-Rh levels in Rpe65/mice gavaged 9-cis-retinal and ERG responses after a long term treatment with 9-cis-retinal. A, comparison of iso-Rh levels in 1-month-old Rpe65−/− mice gavaged with a single dose of 9-cis-retinal (2.5 mg) and kept under 12 h light/dark or at constant dark for 37 days (n = 4). B, the levels of Rh or iso-Rh in 6-month-old Rpe65−/− mice. The Rh levels in wild type mice (column a) were compared with iso-Rh in Rpe65−/− mice treated twice with 9-cis-retinal (2.5 mg each time) at 1 month old with 4-day intervals (column c) and treated twice with 3-month (column d) or 4-month (column e) intervals. No Rh or iso-Rh was detected in untreated Rpe65−/− mice (column b) (n = 4). C, the intensity-dependent response of flicker ERGs in Rpe65+/+, Rpe65−/−, Rpe65−/− treated with 9-cis-retinal, and Rpe65−/− Rgr−/− mice. The flicker recordings were obtained with a range of intensities of 0.00040–41 cd·s/m2 at a fixed frequency (10 Hz). Left panel, Rpe65+/+ mice; right panel, Rpe65−/− with or without treatment (open and closed circles, respectively) and Rpe65−/− Rgr−/− mice without treatment (closed triangles).
F<sc>ig</sc>. 3
Fig. 3
Isomerization, dehydrogenase activity, and phosphorylation of Rh in Rpe65 mice. A, isomerization of all-trans-retinol in RPE microsomes from wild type and Rpe65−/− mice. B, 11-cis-RDH activity in RPE microsomes from wild type and Rpe65−/− mice in the presence of different combinations of dinucleotides. C, immunolabeling of the Rpe mouse retina with monoclonal antibody anti-Rh A11–82P against phosphorylated Rh. Panel A, Rpe65+/+ at constant dark. ROS showed no labeling. Panel B, Rpe65−/− at constant dark. ROS were strongly labeled. Panel C, gavage 9-cis-retinal Rpe65−/− at constant dark without labeling. Panel D, ROS of Rpe65+/+ mice 15 min after the flash showed strong labeling. Panel E, gavage 9-cis-retinal Rpe65−/− mice 15 min after the flash. Immunolabeling is heavy throughout the ROS. In all of the sections, secondary antibody used for detection of anti-phosphorylated Rh antibody recognized choroidal blood vessels and anti-phosphorylated opsin antibody-labeled neurofilaments in inner retina. Scale bar, 20 μm. OS, outer segments; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
F<sc>ig</sc>. 4
Fig. 4
Stimulus response families (A) for Rpe65+/+ and Rpe65/mice supplemented with 2.5, 1.25, 0.25, and 0 mg of 9-cis-retinal. Each panel is the mean family for five rods of each type. Flash (10 ms) strengths increased in two-fold steps from the dimmest intensity that was (in equivalent 500 nm photons/μm2): Rpe65+/+ (3.12), 2. 50 (10.14), 1.25 (190), 0.25 (192), and 0 (1425) mg of 9-cis-retinal. As determined by HPLC retinoid analysis, 300 ± 25 pmol iso-Rh/eye was formed with 2.5 mg, 109.8 pmol of iso-Rh/eye with 1.25 mg and 85.6 ± 6.2 pmol/eye with a 0.25-mg dose of 9-cis-retinal. B, mean light-sensitive dark current for the same sets of rods in A. The error bars are smaller than the symbols. C, mean linear range responses for same cells in A are scaled to the same peak amplitude and superimposed to illustrate differences in response kinetics.
F<sc>ig</sc>. 5
Fig. 5
Mean stimulus response curves (n = 5) of Rpe65+/+ (squares) and Rpe65−/− mice treated with 2.5 (filled circles), 1.25 (open circles), 0.25 (filled triangles), and 0 (brown circles) mg of 9-cis-retinal. The differences in light sensitivity were evaluated by comparing the half-saturating flash intensity (I0) obtained from fitting the mean data with an equation for exponential saturation (38). ECA=AQE(110αl) where R is the peak amplitude of the response, Rmax is the amplitude of the maximum response, and i is the flash strength in photons/μm2. The solid lines are the exponential saturation function (Equation 3) fitted to data with I0 (equivalent 500 nm photons/μm2): 25 (Rpe65+/+), 164 (2.5), 1995 (1.25), 3929 (0.25), and 3714 (0 mg of 9-cis-retinal). Inset, the kinetics of responses adapted by similar amounts (∼4-fold) by steady background illumination (336 equivalent 500-nm photons/μm2/s, black traces) in a Rpe65+/+ rod and by dark light (free opsin) in rod from Rpe65−/− mouse treated with 1.25 mg of 9-cis-retinal. Each trace is from a single rod and is the mean of 10–20 flashes either 6.25 (wild type) or 910 (Rpe65−/− 1.25 mg of 9-cis-retinal (500 nm photon/μm2/flash).
F<sc>ig</sc>. 6
Fig. 6
Photoisomerization of all-trans-retinal in the eyes of Rpe65−/− and Rpe65−/− Rgr−/− mice. A, Rpe65−/− and Rpe65−/− Rgr−/− mice were exposed to a flash that bleached ∼30–35% of Rh in Rpe65+/+ mice. Light-dependent isomerization that resulted in the production of 11-cis-retinal was observed. Four eyes were analyzed. Note that light converts ∼50% of all-trans-retinal to 11-cis-retinal, where the smaller differences in the chromatogram are a result of higher absorption coefficient for all-trans-retinal compared with 11-cis-retinal. B, identification of retinals in Rpe65−/− mice in retina and RPE layers before and after flash. Eight eyes were analyzed. syn-13-cis-Retinal oxime is indicated by an asterisk. The experiments were done using mice, and the tissue was dissected under dim red illumination.

Similar articles

See all similar articles

Cited by 73 articles

See all "Cited by" articles

Publication types

LinkOut - more resources