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INTRODUCTION |
Leber congenital amaurosis
(LCA)1 is a group of
conditions that cause blindness or severe visual impairment from birth.
All show both rod and cone dysfunction, a negligible (not recordable) electroretinogram (ERG), and nystagmus. They result in early onset retinal dystrophy (1), which over time may be accompanied by pigmentary
changes in the retina, hence "amaurosis" (Greek for darken). LCA is
caused by defects in at least five different genes that disrupt a
variety of different cellular functions (2-6).
In ~12% of all LCA cases the gene for a 65-kDa protein (RPE65) of
retinal pigment epithelium cells (RPE) is disabled (7, 8). RPE65 is
heavily expressed in RPE cells, where it plays an essential role in the
retinoid cycle. This is a set of tightly interconnected events that
involve both photoreceptors and RPE cells. The photoisomerization of
the visual pigment chromophore (11-cis-retinal) produces
all-trans-retinal, which is reduced in the photoreceptor,
transferred to the RPE, converted back to 11-cis-retinal,
and then transferred back to the photoreceptor to regenerate the
original visual pigment (reviewed in Ref. 9). The precise function of
RPE65 in retinoid processing is unknown.
Genetically engineered mice in which the gene for Rpe65 has been
eliminated (Rpe65
/) exhibit changes in retinal
morphology, function, and biochemistry that closely resemble the
changes seen in human LCA patients. Both rod and cone function is
severely disrupted, and the ERG is severely attenuated in
Rpe65/ mice (10, 11). There is also a dramatic
overaccumulation of all-trans-retinyl esters in the RPE
cells in lipid-like droplets (10, 12) and degeneration of the retina.
Thus, the Rpe65/ mouse provides the opportunity to gain
insight into the cellular and molecular origins and consequences of LCA
as well as a means to test different therapeutic strategies.
Here we describe the results of an in-depth study of the changes in
biochemistry and function that occur in Rpe65/ mice and
show how the progression of the disease can be interrupted and the
functional effects reversed by providing a supply of
9-cis-retinal. The goals were to: 1) examine the beneficial
effects of 9-cis-retinal treatment on the progression of the
disease and on photoreceptor function; 2) evaluate using single cell
electrophysiology and ERG recording how 9-cis-retinal
treatment affected rod function and light-driven signals in the retina;
and 3) investigate the biochemical basis for the low level of residual
vision that persists in both LCA patients and Rpe65/ mice.
We find that administration of 9-cis-retinal to
Rpe65/ mice produces and maintains rod photopigment for
more than 6 months in the dark. Early intervention with
9-cis-retinal restores normal rod physiology and
significantly attenuates ester accumulation in the RPE but only
partially improves retinal function as measured by ERG. These studies
demonstrate that pharmacological intervention produces long lasting
preservation of visual function in dark-reared Rpe65/
mice and is a potentially useful therapy for restoring vision in LCA patients.
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MATERIALS AND METHODS |
Animals--
All of the animal experiments employed procedures
approved by the University of Washington Animal Care Committee and
conformed with recommendations of the American Veterinary Medical
Association Panel on Euthanasia. Animals were maintained in complete
darkness, and all of the manipulations were performed under dim red
light employing all Kodak No. 1 Safelight filter (transmittance,
>560 nm). Typically, 2-3-month-old mice were used in all of the
experiments. RPE65-deficient mice were obtained from Dr. M. Redmond
(NEI, National Institutes of Health) and genotyped as described
previously (12, 13). Retinal G protein-coupled receptor-deficient mice
were generated and genotyped as described previously (14). Double knockout Rpe65/ Rgr/ were generated by
cross-breeding single Rpe65/ and Rgr/
mice to genetic homogeneity.
Analyses of Retinoids and Visual Pigments--
All of the
procedures were performed under dim red light as described previously
(10, 15, 16). In addition to previously described methods, retinoid
analysis was performed on an HP 1100 series high pressure liquid
chromatograph (HPLC) equipped with a diode array detector and HP
Chemstation A.07.01 software, allowing identification of retinoid
isomers according to their specific retention time and absorption
maxima. A normal phase column (Beckman Ultrasphere Si 5µ, 4.6 × 250 mm) and an isocratic solvent system of 0.5% ethyl acetate in
hexane (v/v) for 15 min followed by 4% ethyl acetate in hexane for 60 min at a flow rate of 1.4 ml/min at 20 °C (total 75 min) with
detection at 325 nm allowed the separation of 11-cis-,
13-cis-, and all-trans-retinyl esters. In
addition, all of the experimental procedures related to the analysis of dissected mouse eyes, derivatization, and separation of retinoids have
been described previously in detail (10). Rh and iso-Rh measurements
were performed as described previously (16). Typically, two mouse eyes
were used per assay, and the assays were repeated three to six times.
The data are presented with S.E.M.
Light and Electron Microscopy--
Eye cups were prepared by
removing the anterior segment and vitreous. The eyes were collected on
ice at PND 1-28 on a weekly basis. "Thin" sections (1.0 µm) were
stained with Richardson's blue solution (1%) and subjected to light
microscopy. "Ultrathin" sections (0.05 µm) were stained with
uranyl acetate/lead citrate and subjected to electron microscopy.
Preparation of Mouse RPE Microsomes--
Fresh mouse eyes were
enucleated immediately after cervical dislocation or CO2
asphyxiation. The anterior segment, vitreous, and retina were carefully
removed under a microdissecting scope. Typically, 30-40 eyes were
dissected for each preparation. RPE cells were separated by placing 12 dissected eyecups in 400 µl of 10 mM MOPS, pH 7.0, containing 1 µM leupeptin and 1 mM
dithiothreitol and vigorously shaken for 20 min. The eyecups were then
gently brushed with a fine brush to further dislodge the RPE cells. The cell suspension was removed, another aliquot of 400 µl of MOPS buffer
was added, and the eyecups were shaken again for 20 min. The cell
suspensions were combined and subjected to glass-glass homogenization.
The homogenate was centrifuged at 10,000 × g for 10 min, and then the supernatant was centrifuged at 275,000 × g for 1 h. The pellet was then reconstituted in 200 µl of the MOPS buffer and resubjected to glass-glass homogenization.
The total protein concentration (typically 0.5-1 mg/ml) was determined by the Bradford method (39).
Isomerization of All-trans-retinol to 11-cis-Retinol using Mouse
RPE Microsomes--
The assay used for determining isomerization to
11-cis-retinol was reported previously (17). Briefly, 20 µl of bovine serum albumin (final concentration, 1%), 125 µl of 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane, pH
7.5, 10 µl of ATP (1 mM final concentration), 25 µM apo-recombinant CRALBP, 40 µl of RPE microsomes
(typically 25-50 µg of total protein), and 0.5 µl of 4 mM all-trans-retinol in dimethylformamide. The reactions were incubated for 2 h at 37 °C. The reaction was
quenched using 300 µl of MeOH, and the retinoids were extracted with
200 µl of hexane. The mixture was shaken vigorously for 2 min and then centrifuged at 14,000 rpm for 4 min for phase separation. The
upper organic layer was removed, and a 100-µl aliquot was separated
and analyzed using an HP 1100 HPLC (Beckman Ultrasphere Si, 4.6 mm x
250 mm, 1.4 ml/min flow rate using 10% ethyl acetate in hexane)
equipped with HP Chemstation software (version A.07.01).
Preparation of pro-S-[4-3H]NADH and
pro-S-[4-3H]NADPH--
Syntheses of
pro-S-[4-3H]NADH and
pro-S-[4-3H]NADPH were carried out with
L-glutamic dehydrogenase, NAD(P), and
L-[2,3-3H]glutamic acid (PerkinElmer Life
Sciences), as described previously (15, 18).
RDH Assays--
The assays were carried out by monitoring the
production of [15-3H]retinol (reduction of retinal) using
11-cis-retinal and
pro-S-[4-3H]NAD(P)H as dinucleotide substrates
in the presence or absence of NADH (18).
Oral Gavage--
Oral gavage was carried out as described
previously (10).
Intravenous Administration of Retinoids--
The chemicals were
purchased from Sigma/Aldrich unless otherwise specified. Solution A
contained 10 mg of 9-cis-retinal, 75 mg of Cremophor EL, 1 mg of 
-tocopherol, and 0.6 mg of benzoic acid suspended in 1 ml of
lactated Ringer's solution (Baxter). The mixture was vortexed
for 10 min and centrifuged for 10 min at 20,000 × g,
and the concentration of 9-cis-retinal (7.7 mM) was determined spectrophotometrically. Solution B contained 13 mg of
9-cis-retinal, 50 mg of Cremophor EL, 10 mg of
dipalmitoylphosphatidyl choline, and 40 mg of
2-hydroxypropyl-
-cyclodextrin suspended in 1 ml of lactated
Ringer's solution (Baxter). The mixture was vortexed for 10 min
and centrifuged for 10 min at 20,000 × g, and the
concentration of 9-cis-retinal (10 mM) was
determined spectrophotometrically. Solutions A and B (typically, 100 µl) were delivered to the mouse lateral tail vein employing a 1-ml syringe equipped with a 27-gauge needle and a restraint tube.
Single Cell Recordings--
Mice were dark-reared from birth and
sacrificed via cervical dislocation, and the eyes were removed. The
retina was isolated and stored on ice for up to 12 h in
HEPES-buffered Ames' solution (10 mM HEPES, pH adjusted to
7.4 with NaOH). Isolated rods were obtained by shredding a small piece
of retina (roughly 1 mm2) with fine needles in a 160-µl
drop of solution. The drop was then injected into a recording chamber
mounted on the stage of an inverted microscope (Nikon Eclipse) equipped
with an infrared video viewing system and continuously superfused at
2-3 ml/min with bicarbonate-buffered Ames' solution warmed to
37 °C (pH 7.4 when equilibrated with 5% CO2, 95%
O2). The entire dissection was carried out under infrared
illumination using a dissecting microscope equipped with
infrared-visible image converters.
An isolated rod was drawn by suction into a heat-polished, silanized
borosilicate electrode with an opening 1.2-1.5 µm in diameter. The
electrode was filled with HEPES-buffered Ames' solution. The
electrical connections to the bath and suction electrode were made by
NaCl-filled agar bridges that contacted calomel half-cells. Bath
voltage was held at ground by an active clamp circuit (19). Membrane
current collected by the suction electrode was amplified by an Axopatch
200A patch clamp amplifier (Axon Instruments, Foster City, CA),
filtered at 30 Hz (3 dB point) with an 8-pole Bessel low pass filter,
and digitized at 1 kHz.
Light from light-emitting diodes with peak outputs at 470, 570, and 640 nm were combined using a trifurcated fiber optic and focused on the
preparation using a water immersion lens in place of the microscope
condensor. The light stimulus was spatially uniform and illuminated a
circular area 0.57 mm in diameter centered on the recorded cell. Light
intensities were measured at the preparation and converted to
equivalent 500-nm photons (
max for rod sensitivity)) using the absorption spectrum of rhodopsin and the measured
light-emitting diode spectrum. Equivalent intensities for each
experiment are given in the figure legends.
Mouse Electroretinograms--
Mice were dark-reared from birth
and anesthetized (ketaject/xylaject, 65 mg/kg intraperitoneally), and
the pupils were dilated with tropicamide (1%). A contact lens
electrode was placed on the eye with a drop of methylcellulose and a
ground electrode placed in the ear. ERGs were recorded and analyzed
with the universal testing and electrophysiologic system 3000 (UTAS
E-3000) (LKC Technologies Inc., Gaithersburg, MD). The mice were placed
in a Ganzfield chamber, and flicker recordings were obtained from one
eye. Flicker stimuli had a range of intensities (0.00040-41 cd·s/m2) with a fixed frequency (10 Hz).
Immunocytochemistry--
Rpe65 mice were divided into
five groups: Rpe65/, Rpe65/ that were
gavaged with 9-cis-retinal and kept in the dark,
Rpe65/ that were gavaged with 9-cis-retinal,
exposed to a flash, and kept in the dark for 15 min,
Rpe65+/+ that were kept in the dark, and Rpe65+/+
that were exposed to a flash and kept in the dark for 15 min. For the
flash experiments, dark-adapted mice were subjected to a flash (Sunpak
433D, 1 ms) from a distance of 2 cm. The retinas were fixed in 4%
paraformalydehyde in 0.13 M sodium phosphate, pH 7.4, for
15 h at 4 °C, and the tissues were transferred to 5, 10, or
15% sucrose in 0.13 M sodium phosphate, pH 7.4, for 30 min
each time and stored overnight in 20% sucrose in the same buffer at
4 °C. The tissue was then transferred to optimal cutting temperature cryoembedding compound and sectioned at 10 µm. The cryosections were incubated overnight at 4 °C in mouse monoclonal anti-phosphorylated Rh A11-82P antibody diluted 1:10. Triton X-100 (0.1%) was included in all phosphate-buffered saline solutions to
facilitate antibody penetration. The controls were processed by
omitting primary antibodies from the incubation buffer. After incubation in primary antibodies, the sections were rinsed with phosphate-buffered saline and then incubated with indocarbocyanine (Cy3)-conjugated goat anti-mouse IgG (1:200). The sections were rinsed
in phosphate-buffered saline mounted in 5% n-propylgallate in glycerol and coverslipped.
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RESULTS |
Early Treatment with 9-cis-Retinal Eliminates Oil-like Structures
in Rpe65/ Mice--
In addition to the loss of photoreceptors, a
defective interface between ROS and RPE, RPE cells of
Rpe65/ mice contained numerous lipid-like droplets (10,
12, 20). In young animals, empty vacuoles were observed in fixed
electron microscopy sections of RPE from Rpe65/ mice but
not in controls (data not shown). With increasing age (>PND 21), they
were filled with a diffractive material that was retained during
electron microscopy section preparation. This observation correlates
with the excessive accumulation of all-trans-retinyl esters
in Rpe65/ mice (Fig.
1A, open circles). Retinyl esters also accumulated with age in Rpe65+/+ mice,
albeit at lower levels than for Rpe65/ mice. By PND 21, ~800 pmol/eye of retinyl esters accumulated compared with ~40
pmol/eye for Rpe65+/+. For Rpe65+/+ mice, Rh
levels initially exceeded the amount of retinyl esters
several-fold.
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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.
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When PND 7 mice were treated with a 0.25-mg dose of
9-cis-retinal (10 mg/ml) every other day until they were 30 days old, a dramatic change in the ester accumulation was observed
(Fig. 1B). With increasing age and continued administration
of 1 dose (1.25 mg) per week, the amounts of
all-trans-retinyl esters increased, similar to
Rpe65+/+, but the overall amounts of esters were
dramatically suppressed with concomitant formation of iso-Rh (Fig.
1C, left panel). Once deposited, the accumulated
esters in the RPE were not removed if the treatment began after more
than 1 month of age (Fig. 1C, right panel). When
young animals or young adults were treated with
9-cis-retinal, the interface contacts between the RPE
and ROS were improved (Fig. 1D, panels d,
f, and h), and the vacuoles appeared to be only
partially filled (Fig. 1D, panels c and
e) over several months of this study. These observations suggest that formation of the regenerated pigment significantly slowed
down accumulation of esters but did not promote the complete removal of
the all-trans-retinyl esters that had been deposited in the eye.
Long Term Effect of 9-cis-Retinal Treatment--
Treatment of mice
with 9-cis-retinal produced a long lasting increase in
photopigment levels and a decrease in accumulation of
all-trans-retinyl esters. Rpe65/ mice
(1-month-old) were treated once (2.5 mg) with 9-cis-retinal
and then kept under either a 12-h light/dark cycle, or under 24 h
dark for 37 days. No appreciable depletion of retinal was observed
under either set of conditions (Fig.
2A). These results suggest
that a single dose of 9-cis-retinal sustains iso-Rh in these
animals under normal laboratory conditions.
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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).
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In another set of experiments, the level of Rh or iso-Rh was measured
in 6-month-old Rpe65/ mice (Fig. 2B). In
these animals, the iso-Rh levels were comparable for three groups of
Rpe65/ mice: mice treated twice with
9-cis-retinal (2.5 mg/dose) at PND 30 and 34, mice treated
twice at PND 30 and 120, and mice treated twice at PND 30 and 150. The
50% decrease of iso-Rh in Rpe65/ (Fig. 2,
B compared with A) matches a similar decrease in
Rh in Rpe65+/+ as a function of age. The ester levels were
reduced by >50% (compared with untreated animals) and were unaffected
by the frequency and dose of 9-cis-retinal. No Rh or iso-Rh
was detected in untreated dark-adapted Rpe65/ mice.
9-cis-Retinal, reduced to 9-cis-retinol, can be
stored in the eye and liver in the form of 9-cis-retinyl
ester. When needed 9-cis-retinol would be liberated by a retinyl
hydrolase. To determine how large the reservoir of
9-cis-retinoids is in the eye and liver, a group of mice
were treated with 9-cis-retinal (2.5 mg) and after 48 h
exposed to multiple flashes at 1-h intervals that bleached ~30-35%
of Rh/flash. Iso-Rh and 9-cis-retinyl esters were
significantly depleted after more than three intense flashes (data not
shown). Retinyl esters from liver and RPE were completely depleted
after five flashes at 24-h intervals (data not shown). Continuous
shedding and resynthesis of Rh-containing ROS discs does not affect the long term preservation of the visual pigment. Therefore, it appears that 9-cis-retinal is, in a large part, recycled from
phagocytized iso-Rh to newly produced opsin molecules over an extended
period of time.
Physiological effects of 9-cis-Retinal Treatment--
Treatment of
Rpe65/ mice with 9-cis-retinal also provided
long term improvement of retinal function. The long term physiological effect of 9-cis-retinal treatment was determined from single
flash responses of different intensities (data not shown) and flicker ERG measurements on Rpe65+/+ and Rpe65/ mice.
Previous experiments showed a partial recovery of the ERG sensitivity
48 h after oral 9-cis-retinal administration (10). We
found that this partial recovery persisted for more than 12 weeks in
Rpe65/ mice treated once at PND 30.
The flicker ERG in Rpe65+/+ mice reached a peak amplitude of
254.9 ± 41.5 µV at a light level of 0.015 cd·s/m2
and 95.1 ± 8.9 µV at 7.5 cd·s/m2 (Fig.
2C, left panel). These data resemble the rod and
cone dominant ERG responses, respectively (11). In
Rpe65/ mice without treatment, the flicker ERG reached a
significantly smaller peak amplitude, 76.0 ± 12.0 µV, at a
light level of 7.5 cd·s/m2 (Fig. 2C,
right panel). Eight weeks after a single treatment with 2.5 mg of 9-cis-retinal, the flicker ERG reached peak amplitudes of 137.3 ± 24.4 µV at 0.059 cd·s/m2 and 40.0 ± 7.1 µV at 13 cd·s/m2 (Fig. 2C,
right panel). These peaks were smaller and occurred at a
higher light level than in the Rpe65+/+ mice; however, the response of treated Rpe65/ mice was 2.1 logarithmic
units more sensitive and had larger amplitude than that of untreated
mice. Thus, administration of 9-cis-retinal provided a long
term, partial recovery of the ERG.
Treatment with 9-cis-Retinal Eliminated Constituitive Opsin
Phosphorylation--
To gain additional insight into the enzymatic
processes of Rpe65 mice, several direct measurements of
relevant enzymatic activities were carried out. It is generally
accepted that opsin has some signaling capability. Immunolabeling on
retina sections from Rpe65 mice using a monoclonal antibody
against phosphorylated opsin could provide a clean evaluation of this
activity, whereas it would be expected that 9-cis-retinal
treatment would inhibit this activity.
The retinas from Rpe65+/+ mice (Fig.
3C, panel A) and
Rpe65/ mice (Fig. 3C, panel B)
were fixed in constant darkness. The ROS in Rpe65+/+ mice
showed no labeling (Fig. 3C, panel A), and the
ROS from untreated Rpe65/ mice were labeled by a
monoclonal antibody against phosphorylated opsin (Fig. 3C,
panel B). This labeling was abolished for
Rpe65/ mice (gavaged once at PND 30 and analyzed 48 h
post-treatment) treated with 9-cis-retinal (Fig.
3C, panel C). This 9-cis-treatment
reduced phosphorylation of opsin to levels comparable with those in
normal rods. ROS fixed in darkness at 15 min following a single flash
showed immunolabeling in both Rpe65+/+ and
Rpe65/ mice treated with 9-cis-retinal (Fig.
3C, panels D and E). These data
suggest that opsin is constitutively phosphorylated in
Rpe65/ mice. These experiments indicated a specific
deficit in conversion of all-trans-retinol to
11-cis-retinol and constitutive opsin phosphorylation but
not in oxidation of 11-cis-retinol to
11-cis-retinal. Constitutive opsin phosphorylation could be
an important element in the pathogenesis of LCA.
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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.
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To directly measure the isomerase activity, RPE microsomes were
isolated from Rpe65 mice using a novel procedure. In control experiments using RPE microsomes from Rpe65+/+ mice,
11-cis-retinol was produced from exogenously added
all-trans-retinol only in the presence of RPE microsomes and
CRALBP (Fig. 3A, upper panel). 11-cis-Retinol was absent when CRALBP was omitted, as well
as when RPE microsomes or CRALBP were denatured by heat.
11-cis-Retinol was not detected in RPE microsomes from
Rpe65/ mice (Fig. 3A, lower
panel).
Because 11-cis-retinol dehydrogenase (11-cis-RDH)
was purified in a complex with RPE65 protein (21), oxidation of
11-cis-retinol was investigated in RPE microsomes from
Rpe65 mice. Strong activity was detected in
Rpe65+/+ and Rpe65/ mice using NADPH and NADH as a dinucleotide cofactor (Fig. 3B). To distinguish
NADPH-dependent activity from NADH-dependent
activity, the test for dehydrogenase activity was carried out in the
presence of nonradioactive NADH and [3H]NADPH. In such
conditions, only NADPH-dependent dehydrogenase activity can
be readily detected (Fig. 3B). The differences between Rpe65+/+ and Rpe65/ were insignificant
because this activity is much higher than required for normal flow of
retinoids as determined from 11-cis-Rdh/ mice
(15). These data suggest that RPE microsomes from Rpe65/
mice contain high NADPH-dependent and
NADH-dependent dehydrogenase activities. In addition, no
differences were seen in immunolocalization of 11-cis-RDH in
the RPE of Rpe65 mice (data not shown).
Treatment with 9-cis-Retinal Restores Normal Rod
Function--
Because the ERG primarily reflects bipolar responses,
the inability of 9-cis-retinal to provide complete recovery
could be due to residual deficits in the photoreceptors or problems in signal transfer from rods to bipolar cells. To determine whether 9-cis-retinal treatment could restore normal photoreceptor
function, we used suction electrodes to record the responses of single
rods from Rpe65+/+ mice and untreated and treated
Rpe65/ mice (gavaged once at PND 30 and analyzed 48 h
post-treatment).
Light-evoked changes in circulating dark current were recorded from
outer segments of single rods from Rpe65+/+ mice or
Rpe65/ mice gavaged 0, 0.25, 1.25, or 2.5 mg of
9-cis-retinal (once a day for two consecutive days preceding
the experiment). Retinoid analysis revealed that 300 ± 25 pmol of
iso-Rh/eye was formed with a 2.5 mg dose of 9-cis-retinal,
109.8 pmol of iso-Rh/eye with a 1.25-mg dose, and 85.6 ± 6.2 of
pmol/eye with a 0.25-mg dose. The nonlinear relation between the dose
of 9-cis-retinal and the iso-Rh concentration presumably
reflects accumulation in the liver and other tissues. All of the rod
types listed supported light responses that increased with increasing
flash strength to reach a maximum (saturating) amplitude when the light
was bright enough to cause all of the cGMP channels to close and fully
suppress the light-sensitive dark current of the cell. The response
families from rods of each type are shown in Fig.
4A. They show that the amplitude of the saturating response increases with increasing doses of
9-cis-retinal. The relationship between mean dark current for each group of rods and the dose of 9-cis-retinal is
plotted in Fig. 4B. Light-sensitive dark current in
Rpe65/ rods that received no supplemental
9-cis-retinal was 2.1 ± 0.3 pA, not significantly different from Rpe65/ rods that received 0.25 mg of
9-cis-retinal (3.6 ± 0.9 pA). Rod dark current
increased with larger doses of chromophore, reaching a value that was
essentially the same as Rpe65+/+ when mice where given 2.5 mg of 9-cis-retinal.
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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.
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Two other properties of the Rpe65/ flash response varied
with the amount of supplemental 9-cis-retinal, response
kinetics, and light sensitivity. To illustrate the kinetic differences, the average dim flash response (in the cells linear range) was determined for each rod type. The mean responses from the five different sets of rods were scaled to the same peak amplitude and are
compared in Fig. 4C. Responses recorded from
Rpe65+/+ and Rpe65/ rods from mice treated
with 2.5 mg of 9-cis-retinal have essentially the same
kinetics. The linear range responses are superimposed, showing that the
dim flash responses of the two different rod types have the same
time-to-peak and recovery times. Responses recorded from rods from
Rpe65/ mice gavaged with 1.25 or 0.25 mg of
9-cis-retinal are also essentially the same, with similar
time-to-peak and recovery times; both are substantially faster than
those of Rpe65+/+ (Fig. 4C, red and
green traces). The dim flash kinetics of rod responses from
Rpe65/ mice that received no supplemental
9-cis-retinal (Fig. 4C, brown trace) were intermediate; they were faster than Rpe65+/+ but slower
than rods from mice treated with 1.25 or 0.25 mg of
9-cis-retinal.
The differences in light sensitivity between Rpe65+/+ rods
and rods from Rpe65/ mice are shown in Fig.
5, which plots the stimulus response
curves for each of the five experimental conditions (Rpe65+/+ and Rpe65/ mice gavaged with 2.5, 1.25, 0.25, or 0 mg of 9-cis-retinal). The half-saturating
flash intensity was lowest in Rpe65+/+ rods (~30
photons/µm2) and increased by factors of 6, 66, and 131 in rods from mice gavaged with 2.5, 1.25, and 0.25 mg of
9-cis-retinal, respectively. The light sensitivity of rods
from mice that did not receive 9-cis-retinal was the same as
rods from mice that received the lowest dose (0.25 mg).
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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).
|
(Eq. 1)
|
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).
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In the Absence of 9-cis-Retinal Treatment, 11-cis-Retinal Is
Produced in Rpe65/ Mice by
Photoisomerization--
Rpe65/ mice that were never
exposed to light have 11-cis-retinal (identified as oximes)
below detection level in conventional microanalysis of retinoids (10).
However, these mice respond to intense illumination in ERG experiments
(10, 11) and in single cell recordings (current study). To identify
whether 11-cis-retinal is produced by exposure to bright
light, four or eight eyes were used for retinoid analysis instead of
two eyes. For Rpe65/, no significant amounts of
11-cis-retinal were detected for dark-adapted animals (Fig.
6). When more eyes were used for
analysis, less than 0.2 pmol/eye of 11-cis-retinal oximes
were detected in a typical chromatogram. All-trans-retinal
(4.2 ± 1.1 pmol/eye, n = 8) was present, and an
intense flash converted this aldehyde to 2.1 ± 0.6 pmol/eye of
11-cis-retinal (Fig. 6A). The retinoids were
identified by the retention time with authentic standards, and their UV
spectra were measured during the chromatography. Next, it was important
to determine whether photoisomerization resulted from the action of the
"photo-isomerase" retinal G protein-coupled receptor protein.
Double knockout Rpe65/ Rgr/ mice were
generated, and retinoid analyses were carried out. A significant
reduction in free all-trans-retinal was observed (2.2 ± 0.2 pmol/eye), but light flash photo-converted a similar fraction
(~50%) to 11-cis-retinal (Fig. 6). To identify where in
the RPE or in the retina these retinals are present, retina and RPE
were separated and analyzed individually (note that eight eyes were
used). Clearly, the majority of all-trans-retinal was
observed in the retina, whereas 11-cis-retinal was present
mostly in the RPE. Bleaching converted all-trans-retinal to
11-cis-retinal that also resided in the retina (Fig.
6B). Once 11-cis-retinal is formed, its level
does not change after 15, 30, or 120 min in the dark (data not
shown).
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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.
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The ERG analyses of Rpe65/ Rgr/ mice were
not qualitatively different from the responses obtained from
Rpe65/ mice (Fig. 2C, right
panel). Together, these results indicate that there is a retinal
photoisomerization pathway that produces 11-cis-retinal and
regenerates Rh in prior bleached animals.
Different Methods of 9-cis-Retinal Delivery--
An important
point was to compare different ways to deliver
9-cis-retinoids with a goal to not only regenerate iso-Rh
but to also build up reservoirs of cis-retinoids. Two
methods2 were tested: gavage
(as described previously (10)) and intravenous injections. Intravenous
injection is an efficient way of delivering retinoids, and there were
no major differences between aldehyde and alcohol forms or their
isomeric compositions (11-cis versus 9-cis) of cis-retinoids (data not shown).
Intravenous injection of 9-cis-retinal produced iso-Rh when
delivered with and without cyclodextrins (data not shown). Retinal was
cleared out rapidly from the blood but could be stabilized in the
circulation for a longer time in the presence of cyclodextrins
(t1/2 = 12 h versus 23 h) (data
not shown). The addition of cyclodextrin, possibly by extending the
time of circulation, also led to higher accumulation of
9-cis-retinyl esters in the liver or RPE (data not shown). A
rapid clearance of 9-cis-retinal from the bloodstream makes
it necessary to give multiple intravenous injections to fully
regenerate iso-Rh. This is not the case with gavage, in which the
presence of retinal in the bloodstream lasts for >48 h. Together,
gavage and intravenous injections were effective in producing iso-Rh in
Rpe65/ mice. The advantages and disadvantages of both
methods are described under "Discussion."
| |
DISCUSSION |
The Role of RPE65 and LCA--
Although the sequence of events
that lead to the diseased state in Rpe65/ mice, the
animal model of LCA, has not been established, it is likely that the
primary defect is an interruption of the retinoid cycle. This cycle is
responsible for regenerating the visual pigment through the enzymatic
conversion of all-trans-retinal to 11-cis-retinal
in the RPE and its return to the photoreceptor cell. Disruption of the
normal retinoid flow between the RPE and photoreceptor can explain the
overaccumulation of retinal esters in the RPE. Furthermore, the failure
to regenerate rhodopsin can account for diminished rod and cone light
sensitivity (23, 24). The absence of 11-cis-retinal also
increases free opsin in the photoreceptor. A high level of free opsin
produces substantial activation of the phototransduction cascade (Figs.
3C, 4, and 5), mimicking the effects of continuous light
exposure. This ongoing activity may cause the reduction in the
thickness of the ROS layer and photoreceptor degeneration, effects also
produced in animals exposed to continuous light (25-28). This sequence
of events may be further aggravated by the phosphorylation of free
opsin, which has been shown in other studies to lead to retinal
degeneration (29, 30).
Early treatment of Rpe65/ mice with
9-cis-retinal inhibited the accumulation of
all-trans-retinal, improved the attachment contacts between
RPE processes and ROS, led to dephosphorylation of opsin (Figs.
1D and 3C), and prevented the further progression of retinal degeneration. These observations suggest that ester accumulation in the RPE and the presence of high levels of active opsin
in the photoreceptor may be the principle causes of retinal degeneration in the Rpe65/ mouse.
Rescued Rod Function--
The light sensitivity of rods from
Rpe65/ mice was restored in a dose-dependent
manner by dietary supplemental 9-cis-retinal. The highest
dose supported rod responses with normal sensitivity and kinetics (Fig.
4). Treatment with lower doses of 9-cis-retinal gave rise to
rod responses that were desensitized and had faster kinetics, closely
resembling the changes in sensitivity and kinetics that occur during
steady background illumination in wild type rods. The changes in the
light sensitivity of rod responses recorded from mice treated with the
lower amounts of 9-cis-retinal could be accounted for
by a combination of two factors. One source of desensitization was a
decrease in the effective collecting area of the rod because of a
reduction in both the amount of visual pigment and its quantum
efficiency; the quantum efficiency of iso-Rh is about one-third that of
Rh. The remaining reduction in sensitivity could be explained by steady
activation of the transduction cascade by free opsin, producing an
effect equivalent to that caused by steady background illumination in
wild type rods.
Rods from Rpe65/ mice that were not treated with
9-cis-retinal also generated light responses that were
strongly desensitized. The presence of residual rod responses in
untreated Rpe65/ mice is consistent with previous
reports of reduced but present light responses in children with LCA.
Our results indicate that under these conditions the generation of
light responses by flashes of intense light is most likely due to the
production of 11-cis-retinal from the photoconversion of
all-trans-retinal in the retina (Fig. 6). It is open to
speculation whether all-trans-retinal is free or coupled
(either covalently or noncovalently) to opsin. The preassociation of
the chromophore and opsin would make the formation of the
light-sensitive 11-cis-retinal complex (i.e. Rh)
fast enough for it to be subsequently photoisomerized and
transduction-triggered within the period of a brief (10 ms) flash of light.
Phototransduction in Rods of Rpe65 Mice--
The shifts in light
sensitivity rods from treated and untreated Rpe65 mice can
be attributed to a decrease in the effective collecting area of the rod
acting either alone (2.5 mg of 9-cis-retinal) or in addition
to desensitization by an "equivalent background" (31) because of a
low level of steady activation of the transduction cascade by free
opsin (32).
The effective collecting area (ECA) depends on the geometric
collecting area of the rod (A), the quantum efficiency of
the pigment (QE), and the pigment density ().
|
(Eq. 2)
|
where l is the path length. The pigment regenerated
using 9-cis-retinal is iso-Rh, which has about one-third of
the quantum efficiency of Rh (0.22 versus 0.67). The
biochemical measurements indicate that in mice gavaged with 2.5 mg of
9-cis-retinal, all of the pigment is iso-Rh (no free
opsin ± 10%) and is about 57% of the amount of rhodopsin in
Rpe65+/+ rods (i.e. 300 pM iso-Rh versus 525 pM Rh). The decreases in quantum
efficiency and axial pigment density would be expected to cause
~5-fold decrease in effective collecting area of rods from mice fed
with 2.5 mg of 9-cis-retinal. This is in agreement with the
6-fold increase in half-saturating flash strength in rods from
Rpe65/ mice gavaged with 2.5 mg of
9-cis-retinal, compared with rods of Rpe65+/+. In
rods from mice treated with 1.25 and 0.25 mg of
9-cis-retinal, the axial densities of iso-Rh were 21 and
16%, respectively, of the amount of Rh in Rpe65+/+ rods. By
the same reasoning as above, these changes would be expected to
increase the half-saturating flash strength by 14.5- and 19-fold
compared with Rpe65+/+. This is not enough to account for
the observed shifts in sensitivity; rods from mice gavaged with 1.25 and 0.25 mg of 9-cis-retinal are further desensitized by
factors of 4.5- and 6.8-fold, respectively.
The additional desensitization could be attributed to an equivalent
background (31) that acts like "dark light" to cause steady
activation of the cascade. In separate experiments on
Rpe65+/+ rods the change in flash sensitivity by background
illumination was described by the Weber-Fechner relationship.
|
(Eq. 3)
|
where Sf is the flash sensitivity in steady
light, S
is flash
sensitivity in darkness, Ib is the background light intensity, and I
is
the background intensity (108 photons/µm2/s)
that reduces the flash sensitivity by half its dark value. Hence,
background intensities of 378 and 648 photons/µm2/s would
be expected to cause 4.5- and 7-fold changes in flash sensitivity. With
an effective collecting area of 0.5 µm2 and an
integration time of 0.3 s, these background intensities correspond
to equivalent activation in Rpe65+/+ rods of 57 and 97 Rh*/s.
We determined the equivalent background of residual free opsin in the
treated Rpe65/ rods by combining biochemical
measurements of the free opsin concentration with physiological
estimates of desensitization. The number of Rh molecules in a
Rpe65+/+ rod is estimated to be about 2 × 107 (i.e. 3 mM Rh in 0.02 pl).
Biochemical measurements on rods from Rpe65/ mice
indicate that they make ~40% less pigment than Rpe65+/+ 48 h after treatment. Thus, the number of iso-Rh molecules in rods
from Rpe65/ mice gavaged with 2.5 mg of
9-cis-retinal would be about 1.2 × 107.
Smaller doses of 9-cis-retinal do not regenerate all of the available pigment to form iso-Rh, causing there to be a pool of free
opsin. The retinoid analysis suggests that the amount of free opsin in
rods from Rpe65/ mice gavaged with 1.25 and 0.25 mg of
9-cis-retinal would be 63 and 72% of the total amount of available pigment (i.e. 7.5-8.6 × 106
molecules). For this amount of free opsin to cause desensitization in
the rods from Rpe65/ mice that is equivalent to the
desensitization in Rpe65+/+ rods caused by a steady light
that bleaches 57 and 97 Rh*/s, about 1 × 105 opsin
would have to activate the cascade as well as 1 Rh* (1.3-0.9 × 105 opsin: Rh*). This value is broadly consistent with
previous estimates of activation ratio of free opsin: Rh*
(i.e. 106:1) (33). The inset in Fig.
5 shows that background light adaptation and adaptation by an
equivalent (free opsin) background that desensitized the flash response
by similar amounts had similar effects on the kinetics of the dim flash
response. This is also in general agreement with previous studies (32)
that showed the adaptational changes in the kinetics of the dim flash
response were similar, whether adaptation was due to background light
or the equivalent background associated with dark adaptation.
The highly desensitized rod responses recorded from untreated
Rpe65/ mice did not show the acceleration in response
kinetics seen in rods from treated mice (Fig. 4C). There are
several possible explanations for this difference. One possibility is
that the activity of free opsin is less in rods from untreated
Rpe65/ mice than in those from treated mice, perhaps
because of phosphorylation of the opsin in untreated rods. This
explanation would require that treatment with a low dose of
9-cis-retinal converts most or all of the remaining free
opsin to a state of higher activity, perhaps through dephosphorylation.
Another possibility is that the activation and deactivation of the
photopigment are altered in the untreated mice. For example, it is not
clear that the photopigment created by photoconversion is identical to
normal rhodopsin; for example, the opsin may still be phosphorylated.
Further studies are required to distinguish these possibilities.
The complete or nearly complete rescue of normal rod function after
treatment with 9-cis-retinal contrasted with the partial rescue of the sensitivity of the electroretinogram. Because the electroretinogram primarily reflects activity of bipolar cells, this
difference indicates that responses in the rods are not properly transmitted across the rod-bipolar synapse. It is possible this synapse
does not develop properly in Rpe65/ mice because of a
lack of visual signals. Continuous treatment with
9-cis-retinal from birth may help remedy this problem.
Advantages and Disadvantages of 9-cis-Retinal
Treatments--
Retinals can be delivered to the eye effectively by
one (or a combination) of two methods: gavage (10) and intravenous
injection (current study). The most effective delivery system is
gavage, which restores visual pigment in 1-2 days and also produces
accumulation of 9-cis-retinyl esters in the liver and RPE
microsomes. It is a highly reproducible procedure. There is a transient
elevation of retinoids in the blood for 48 h that is followed by
recovery to the normal level. The only noticeable drawback is that much of the retinoid is secreted rather than stored, requiring a higher dose
than other delivery methods.
Intravenous injection is also an effective method for retinoid delivery
to the eye, but it has the disadvantage of the retinoids being rapidly
eliminated from the bloodstream by the kidneys. This can be prevented
to some degree by "caging" retinal in a cyclodextrin net. For full
regeneration, multiple or large doses must be injected, causing
potential problems with local infection. To lower the amounts of
circulating all-trans-retinoids, it would be helpful to
inhibit liver carboxylesterase to prevent all-trans-retinal from being released to the bloodstream. Such inhibitors, if they are
potent, are highly toxic, because they inhibit other processes that
require hydrolase activity. General and mild inhibitors, such as
vitamins K1 and E (34), are effective to some
degree,3 but more specific
inhibitors would be useful to enhance the level of
cis-retinoids in the bloodstream. Finally, intraocular
injection (22) is an option in same cases. It requires the smallest
amounts of material and is useful for specialized retinoids, but this is not a readily accessible or routine procedure, and repeated intraocular injections are associated with the formation of cataracts (20-40%).
There is not a large reservoir of cis-retinoids in the liver
and RPE, most likely because of nonenzymatic conversion of free retinal
or retinol to the all-trans isomer (22). However, the efficiency of mammalian vision is remarkable and worth consideration in
light of potential cis-retinoid therapy. For example, the
mammalian retina contains ~108 photoreceptors. If each
photoreceptor absorbs on average 1-2 × 103
photons/s, with a quantum yield of 0.65 (or 0.3 for
9-cis-retinal), the daily requirement of
11-cis-retinal is only <1 µg, an amount that could be
easily delivered by dietary supplement even if the majority of
retinoids are retained in liver or secreted. The recommendations for
vitamin A intake is 0.8 mg/day for men and 0.7 mg/day for women, with
the upper safety limit of 3 mg/day is only an estimate, because of lack
of data (35).
Multiple gavages do not increase the amount of retinyl esters in the
eye. In contrast, early intervention significantly lowers the
accumulation of all-trans-retinyl esters (Fig. 1). This
could be one of the prerequisites of successful cis-retinoid
therapy for retinal diseases. The level of all-trans-retinyl
esters in the RPE is predetermined by the time of the intervention. If
the treatment is initiated very early in life, the esters only
gradually increase with age, as in wild type mice. The treatment does
not remove the esters from the eye but prevents accumulation of the esters. One possible explanation is that the retina sends a signal that
opsin is not regenerated, and this causes retinol capture from the
blood circulation and retention as retinyl ester in RPE. When retinyl
esters cannot be converted to 11-cis-retinal, and the
"opsin signal" is on, these two factors ultimately lead to ester
accumulation. The mechanism of such communication is unknown on a
molecular level.
In all of the experiments described here, we did not observe any
adverse effects of 9-cis-retinal. However, in applying these results to other species, it is important to note that retinoid flow in
the mouse may be substantially different from in other animals. For
example, dogs have very high levels of retinoids in the bloodstream
(36, 37) that will need to be combated to efficiently deliver
cis-retinoids to the eye via gavage.
In summary, we provide evidence that administration of
9-cis-retinal restores rod photopigment and rod retinal
function for more than 6 months and that early intervention
significantly attenuates the ester accumulation. Opsin in
Rpe65/ mice is constitutively phosphorylated in rods of
Rpe65/ mice, and this modification of the visual pigment
could be involved in the pathophysiology of LCA; fortunately, after
9-cis-retinal-treatment, opsin is dephosphorylated. We also
provide evidence that the source of 11-cis-retinal in Rpe65/ mice results from photoisomerization of
all-trans-retinal present in the retina and that other
mechanisms in addition to photoisomerase retinal G protein-coupled
receptor are involved in this process, as shown in double
Rpe65/ Rgr/ knockout mice. Electrophysiological data using single cell recordings suggest that
11-cis-retinal is formed in situ in rod outer segments.
These studies provide information about the etiology of LCA on a
molecular level and demonstrate that pharmacological intervention
produces long lasting preservation of the visual function in
dark-reared Rpe65/ mice.
,
,
TOP
ABSTRACT
INTRODUCTION
