October 2010
Volume 51, Issue 10
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Retina  |   October 2010
Retinal Disease in Rpe65-Deficient Mice: Comparison to Human Leber Congenital Amaurosis Due to RPE65 Mutations
Author Affiliations & Notes
  • Rafael C. Caruso
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Tomas S. Aleman
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Artur V. Cideciyan
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Alejandro J. Roman
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Alexander Sumaroka
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Cristina L. Mullins
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Sanford L. Boye
    the Department of Ophthalmology, University of Florida, Gainesville, Florida.
  • William W. Hauswirth
    the Department of Ophthalmology, University of Florida, Gainesville, Florida.
  • Samuel G. Jacobson
    From the Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Corresponding author: Samuel G. Jacobson, Scheie Eye Institute, University of Pennsylvania, 51 N. 39th Street, Philadelphia, PA 19104; jacobsos@mail.med.upenn.edu
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science October 2010, Vol.51, 5304-5313. doi:10.1167/iovs.10-5559
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      Rafael C. Caruso, Tomas S. Aleman, Artur V. Cideciyan, Alejandro J. Roman, Alexander Sumaroka, Cristina L. Mullins, Sanford L. Boye, William W. Hauswirth, Samuel G. Jacobson; Retinal Disease in Rpe65-Deficient Mice: Comparison to Human Leber Congenital Amaurosis Due to RPE65 Mutations. Invest. Ophthalmol. Vis. Sci. 2010;51(10):5304-5313. doi: 10.1167/iovs.10-5559.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To quantify the retinal disease in Rpe65-deficient mice across a wide age span and compare the results to those in humans with Leber congenital amaurosis (LCA) caused by RPE65 mutations.

Methods.: Full-field electroretinograms (ERGs) were recorded from wild-type (C57BL/6; Rpe65 +/+) and Rpe65 −/− mice at ages ranging from ∼1 month to 2 years. A physiologically based model of rod phototransduction activation was used to determine photoreceptor (P3) cell components of ERG photoresponses. A bipolar (P2) cell component was also derived. Photoreceptor and inner retinal thickness measurements were made by using optical coherence tomography in human RPE65-LCA.

Results.: Age-related declines in ERG photoreceptor and bipolar amplitudes were present in the Rpe65 −/− mouse. The loss of photoresponse amplitude with age in the mutant mice paralleled reported losses of photoreceptor nuclear layer thickness over the same age range. Unexpectedly, the early activation phase of photoresponses in Rpe65 −/− mice accelerated with age as amplitude decreased; this was not a feature of Rpe65 +/+ mice. Inner retinal dysfunction increased with age in the mutant mice. Human RPE65-LCA patients had retinal degeneration and loss of photoreceptors in the first decade of life. Unlike the mouse model, there were no examples of a normal photoreceptor complement. Abnormal thickening of the inner retina occurred with increasing loss of photoreceptors.

Conclusions.: The differences in time course of murine and human RPE65-deficiency diseases suggests that preclinical efficacy testing of therapeutic modalities would be most informative when the murine disease becomes comparable to early human disease, toward the end of the first year of life in Rpe65 −/− mice.

Leber congenital amaurosis (LCA) is a group of childhood-onset human retinal diseases known to be caused by mutations in at least 14 different genes, with many different disease mechanisms. 1,2 One of the most explored and widely publicized forms of LCA is due to mutations in the gene encoding RPE65 (retinal pigment epithelium–specific-65-kDa), the isomerase of the visual cycle. 3 Treatment of this form of LCA has recently been shown to be safe and efficacious in independent early-phase clinical trials of subretinal injection of adeno-associated viral vector delivery systems. 37  
Studies of canine and murine animal models with RPE65 deficiency have had a major positive impact on the relatively rapid translation to human clinical trials of this gene therapy (reviewed in Refs. 2, 3). Differences between the models and the human disease have been observed, 810 and such results prompt the need for caution in extrapolation from one to the other. Yet, there is also the opportunity in the animals to understand more about the natural history of the disease as it converts from a relatively pure biochemical dysfunction to a combined retinal dysfunction and degeneration. 
We studied the time course of disease in one of the most commonly used murine models of Rpe65 deficiency, the Rpe65 −/− mouse. 11 Photoreceptor function over much of the lifetime of the animal model was quantified, and whether loss of function has a relation to underlying retinal degeneration was determined. Whether inner retinal dysfunction contributes to the visual loss was also explored. Comparisons were made between the disease course in Rpe65-deficient mice and in humans with RPE65-LCA. 
Methods
Animals
C57BL/6 wild-type mice (Rpe65 +/+, n = 42; age range, 1.1–27.2 months) served as control animals. Rpe65 −/− mice bred on a C57BL/6 background were included in the study (n = 57; age range, 1.1–28.5 months). The animals were raised from birth in 12-hour-on/12-hour-off cyclic lighting (ambient illumination <3 lux). Access to food and water was ad libitum. Procedures were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with institutional approval. 
Electroretinography
Full-field ERGs were performed as previously described 12,13 by using a custom-built Ganzfeld, a computer-based system (EPIC-XL; LKC Technologies, Gaithersburg, MD), and specially made contact lens electrodes (Hansen Ophthalmics, Iowa City, IA). The mice were dark-adapted (>12 hours) and anesthetized by intramuscular injection of a mixture of ketamine HCl (65 mg/kg) and xylazine (5 mg/kg). The pupils were dilated with tropicamide (1%) and phenylephrine (2.5%). Medium-energy (10-μs duration) and high-energy (1-ms duration) flash stimulators were used; unattenuated luminances were 0.8 and 3.6 log scot-cd · s · m−2, respectively (all references to log throughout the manuscript refer to common logarithm to the base 10). Neutral density (Wratten 96) and blue (Wratten 47A; Eastman Kodak, Rochester, NY) filters were used to control the intensity and spectral composition of the stimulus. Signals evoked by medium-energy flashes were amplified, filtered (−3-dB cutoff at 0.3 and 300 Hz), and digitized (2 kHz) with a 12-bit analog-to-digital converter. Signals from higher energy flashes were recorded with higher bandwidth (1500-Hz filtering and 3.33-kHz sampling). The study protocol began with dark-adapted ERGs elicited with increasing intensities of blue light flashes from −4.2 to 0.1 log scot-cd · s · m−2. 13 After a 2-minute wait, an ERG photoresponse was evoked with a single, blue, 2.2-log scot-cd · s · m−2 flash. A single, white, 3.6-log scot-cd · s · m−2 flash was used after a 2-minute wait, to evoke the maximum photoresponse. 
Phototransduction Activation Phase Modeling
Leading edges (4–20 ms, depending on the response) of dark-adapted ERG a-waves (ERG photoresponses) evoked with the 2.2- and 3.6-log scot-cd · s · m−2 flashes were fit with a physiologically based model of rod phototransduction activation, 1217 as they are thought to represent the retina-wide sum of light-induced dark-current shutoff in mouse rod photoreceptor outer segments. 18 Maximum amplitude (P3max) and sensitivity (P3sens) parameters were derived by minimizing the average root-mean-square error with a simplex algorithm, while holding the remaining parameters 14 constant (τsc = 0.85 ms; τOS = 0.5 ms; δ = 1.1 ms); the fastest photoresponse was assumed to have a saturated amplitude. It is important to note that the rigor in estimating the underlying phototransduction parameters is significantly increased by requiring a single set of physiologically based model parameters to describe ERG data covering 1.5 log units of dynamic range, and this is especially important for the smaller signals in Rpe65 −/− mice. 
When isolated from potentials believed to originate in more distal or more proximal retinal cells, the ERG b-wave is called the P2 component. 19 We derived the P2 component of the ERG at maximum illumination (3.6 log scot-cd · s · m−2) by subtracting the P3 component model from the ERG data. The amplitude was measured at the fixed time of 80 ms (P280), as described elsewhere. 12  
Human Subjects
Nineteen patients (ages, 7–40 years) with LCA caused by mutations in the RPE65 gene 810,20 were included in the study. They underwent a complete eye examination and optical coherence tomography (OCT). Normal subjects for optical coherence tomography (n = 28; ages, 5–58 years) were also included. Informed consent was obtained for all subjects; procedures adhered to the Declaration of Helsinki and were approved by the institutional review board. 
Optical Coherence Tomography
Retinal cross sections were obtained by OCT; the principles of the method and our recording and analysis techniques have been published. 8,2123 Some of the data were acquired with an OCT3 instrument (Carl Zeiss Meditec, Inc., Dublin, CA), whereas other data were obtained with Fourier-domain (FD) OCT imaging (RTVue-100; Optovue, Inc., Fremont, CA). Dense raster scans were performed to sample an 18 × 12-mm region of the retina centered on the fovea. A video fundus image was acquired with each OCT scan. The location and orientation of the scan relative to retinal features were determined by using the digital images of the fundus. 
Postacquisition processing of OCT data was performed with custom programs written in commercial software (MatLab 6.5; MathWorks, Natick, MA). Longitudinal reflectivity profiles (LRPs) making up the OCT scans were aligned by straightening the major RPE reflection. 8,2123 The outer photoreceptor nuclear layer (ONL) and inner nuclear layer (INL) were defined in regions of the scans showing two parallel stereotypical hyporeflective layers between the RPE and vitreoretinal interface. 8,23 Inner retinal thickness was defined as the distance between the signal transition at the vitreoretinal interface and the sclerad boundary of the INL or the single hyporeflective layer continuous with the INL. 8,23 In normal subjects, the signal corresponding to the RPE was assumed to be the most sclerad peak within the multipeaked scattering signal complex, 21 deep in the retina. In abnormal retinas, the presumed RPE peak was sometimes the only signal peak deep in the retina; in other cases, it was apposed by other major peaks. In the latter case, the RPE peak was specified manually by considering the properties of the backscattering signal originating from layers vitread and sclerad to it. 24  
For topographic analysis, the precise location and orientation of each scan relative to retinal features (blood vessels, intraretinal pigment, and optic nerve head) were determined by using the video images of the fundus. LRPs were allotted to regularly spaced bins (0.3 × 0.3 mm) in a rectangular coordinate system centered at the fovea; the waveforms in each bin were aligned and averaged. Overall, ONL and inner retinal thickness were measured as described earlier. Missing data were interpolated bilinearly, thicknesses were mapped to a pseudocolor scale, and locations of blood vessels and optic nerve head were overlaid for reference. 25 For ONL thickness analysis, the foveal region (2 × 2 mm centered at the fovea), 26 as well as a 3 × 3-mm region centered on the optic nerve head, were excluded. For an inner retinal thickness versus ONL thickness comparison, the analysis focused on the temporal retina away from the main nerve fiber layer bundles. Measurements were made in the region extending from 2 to 8 mm in the temporal retina and superiorly and inferiorly (up to 4.8 mm of eccentricity). Data from normal subjects (n = 3, ages, 24, 26, and 43 years; ∼200 loci per subject) were used to generate average ONL and inner retinal thickness maps. Both inner retinal and ONL thicknesses were specified as a fraction (in percentage) or change (in microns) from the mean normal thickness calculated at each retinal location. 
Results
Changes in Retinal Function with Age in Rpe65−/− Mice
ERG waveforms elicited in the dark-adapted state in response to increasing stimulus light intensities are shown for Rpe65 +/+ and Rpe65 −/− mice of two ages: 3 and 9 months (Fig. 1A). The representative ERGs from the Rpe65 +/+ mouse show that at lower stimulus intensities, there was a threshold for the b-wave and with further increases in intensity, b-wave amplitude became larger and a-waves were visible. Inspection of the waveforms for the older Rpe65 +/+ animal indicated that, compared with those of the younger mouse, there were slightly lower amplitude responses to the stimuli presented. 
Figure 1.
 
Retinal function change with age in the Rpe65 +/+ and the Rpe65 −/− mice. (A) ERG luminance-response series obtained in dark-adapted conditions over a range from −4.2 to 3.6 log scot-cd · s · m−2 flashes show elevation of b-wave thresholds in the Rpe65 −/− mice by ∼3 log units compared with those in the Rpe65 +/+ mice at the ages of 3 and 9 months (m). ERG amplitude responses to the highest intensities of light were reduced in the older mouse compared with those in the 3-month-old. All traces start at flash onset. (B) Leading edges of dark-adapted ERG photoresponses evoked with 2.2- and 3.6-log-scot-cd · s · m−2 flashes (thin traces) fit as an ensemble with a model of rod phototransduction activation (thick traces). Representative results are shown for two ages. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (C) Rod photoreceptor function as a function of age estimated with the P3max parameter over the ages from 1 to 25 months in the Rpe65 +/+ (left) and the Rpe65 −/− (right) mice. Regression lines (thick gray) describe log-linear change in the parameters with age; 95% prediction intervals (thin gray lines) encompassing the data are also shown. For Rpe65 −/− mice, only data between 1 and 16 months of age were used for regression analyses, to avoid the uncertainty resulting from near-noise-level responses at ages older than 20 months. (D) Bipolar cell function as a function of age estimated with the P280 parameter with analyses comparable to those in (C).
Figure 1.
 
Retinal function change with age in the Rpe65 +/+ and the Rpe65 −/− mice. (A) ERG luminance-response series obtained in dark-adapted conditions over a range from −4.2 to 3.6 log scot-cd · s · m−2 flashes show elevation of b-wave thresholds in the Rpe65 −/− mice by ∼3 log units compared with those in the Rpe65 +/+ mice at the ages of 3 and 9 months (m). ERG amplitude responses to the highest intensities of light were reduced in the older mouse compared with those in the 3-month-old. All traces start at flash onset. (B) Leading edges of dark-adapted ERG photoresponses evoked with 2.2- and 3.6-log-scot-cd · s · m−2 flashes (thin traces) fit as an ensemble with a model of rod phototransduction activation (thick traces). Representative results are shown for two ages. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (C) Rod photoreceptor function as a function of age estimated with the P3max parameter over the ages from 1 to 25 months in the Rpe65 +/+ (left) and the Rpe65 −/− (right) mice. Regression lines (thick gray) describe log-linear change in the parameters with age; 95% prediction intervals (thin gray lines) encompassing the data are also shown. For Rpe65 −/− mice, only data between 1 and 16 months of age were used for regression analyses, to avoid the uncertainty resulting from near-noise-level responses at ages older than 20 months. (D) Bipolar cell function as a function of age estimated with the P280 parameter with analyses comparable to those in (C).
The Rpe65 −/− mice at 3 and 9 months of age had b-wave thresholds that were very elevated and amplitudes that were reduced compared with those in the Rpe65 +/+ mice of the same ages (Fig. 1A). Dim light stimuli that would be expected to elicit rod-mediated responses in Rpe65 +/+ mice (−4.2 to −1.2 log scot-cd · s · m−2) elicited no detectable waveforms in the Rpe65 −/− mice. The older mutant mouse had lower amplitudes than did the younger mouse. 
The leading edges of the dark-adapted ERG photoresponses are well fit with the alternative model of rod phototransduction activation (Fig. 1B). Representative results show that the Rpe65 −/− mice had a smaller P3max than did the Rpe65 +/+ mice at both ages (406.7 vs. 77.4 μV at 3 months; 315.5 vs. 32.7 μV at 9 months). ERG data for all Rpe65 +/+ and Rpe65 −/− mice in the study were analyzed and plotted as a function of age (Figs. 1C, 1D). The photoreceptor component amplitude in Rpe65 +/+ mice declined with age (Fig. 1C, left) and can be described with a log-linear function: log(P3max) = 2.58 − 0.018 · months; r 2 = 0.57. In the Rpe65 −/− mice studied (Fig. 1C, right), P3max amplitude also declined as a function of age. The reduction of amplitude was also consistent with a log-linear function with a more negative slope, log(P3max) = 2.01 − 0.039 · months; r 2 = 0.65, implying a greater age-related loss of photoreceptor function in Rpe65 −/− mice compared with Rpe65 +/+. Bipolar cell function was estimated by the amplitude of the P2 component (P280) evoked with the maximum illumination. Rpe65 +/+ mice showed a decline in bipolar cell function with age (Fig. 1D, left) that could also be well described with a log-linear function: log(P280) = 2.77 − 0.017 · months; r 2 = 0.54); the slope of bipolar cell function decline was essentially equivalent to that of the photoreceptor amplitude decline. In Rpe65 −/− mice, bipolar function also declined and could be described with a log-linear function: log(P280) = 2.24 − 0.025 · months; r 2 = 0.27; however, there were greater variability and limited data above the noise floor at later ages (Fig. 1D, right). 
Retinal Function versus Photoreceptor Layer Structure in Rpe65−/− Mice: Comparison to Human RPE65-LCA
Age-related declines in normal mouse retinal structure have been reported and debated (for example, Refs. 2729), but there is greater consensus that retinal function, as quantified by ERGs, does decline. 28,29 Our measured declines in P3 and P2 component amplitudes in Rpe65 +/+ mice with age (Figs. 1C, 1D) are consistent with reports in the literature. 27,28 The basis of the greater rate of decline in ERG amplitude parameters in Rpe65 −/− mice could be due to any of several mechanisms, such as progressive photoreceptor degeneration, increasing loss of RPE cells, changes in pathways for generation of remnant chromophore, change in the level of spontaneous activity of the free opsin, or a combination of factors. 3032 To test the hypothesis that progressive photoreceptor loss contributes to the decline in ERG, we relied on illustrated retinal histopathology or published measurements of ONL in Rpe65 −/− mice at different ages and the histologic data of normal mice that accompanied the mutant data in some of the studies. 8,11,3236 We plotted, as a function of age, the relationship of ONL thickness in the Rpe65 −/− retina as a percentage of Rpe65 +/+ retina (Fig. 2A). On the same graph as the ONL data, we plotted the photoreceptor function parameter P3max in Rpe65 −/− mice as a percentage of Rpe65 +/+ for the different ages. The similarity in the rates of age-related decline in ONL thickness and photoreceptor function parameters suggest that photoreceptor cell loss is a dominant contributor to the progressive reduction of P3max amplitude in the natural history of Rpe65 −/− disease. 
Figure 2.
 
Photoreceptor nuclear layer thickness loss in Rpe65−/− mice and in humans with RPE65-LCA. (A) Comparison of relative loss of photoreceptor ONL thickness with age to the relative loss of photoreceptor function with age. ONL thicknesses in the Rpe65−/− mice are taken from the literature: (Image not available) Redmond et al.11; (■) Gouras et al.33; (▴) Woodruff et al.32; (▲) Rohrer et al.34; ([Image not available]) Jacobson et al.8; (▾) Fan et al.35; and (♦) Samardzija et al.36 The data were normalized by the thickness of Rpe65+/+ at matched ages, when available. P3max parameter (unfilled symbols) for individual Rpe65−/− mice were divided by the log-linear estimate of Rpe65+/+ natural history at each age. Thick lines: log-linear progression estimates for the relative ONL loss and relative P3max loss. (B) Topographical maps from OCT measurements in patients with RPE65-LCA compared with mean normal. Top left: mean normal map of ONL thickness; middle left, ONL thickness map in a 7-year-old patient with RPE65-LCA; top right: patient ONL compared with mean normal ONL (percentage). Two other ONL percentage maps are shown on the right (12 and 22 years of age). Bottom left: ranking by percentage of ONL across this wide region of central retina in 17 patients of different ages (age of each patient listed below each bar). Left-hatched bar on the y-axis: lower limit of normal range.
Figure 2.
 
Photoreceptor nuclear layer thickness loss in Rpe65−/− mice and in humans with RPE65-LCA. (A) Comparison of relative loss of photoreceptor ONL thickness with age to the relative loss of photoreceptor function with age. ONL thicknesses in the Rpe65−/− mice are taken from the literature: (Image not available) Redmond et al.11; (■) Gouras et al.33; (▴) Woodruff et al.32; (▲) Rohrer et al.34; ([Image not available]) Jacobson et al.8; (▾) Fan et al.35; and (♦) Samardzija et al.36 The data were normalized by the thickness of Rpe65+/+ at matched ages, when available. P3max parameter (unfilled symbols) for individual Rpe65−/− mice were divided by the log-linear estimate of Rpe65+/+ natural history at each age. Thick lines: log-linear progression estimates for the relative ONL loss and relative P3max loss. (B) Topographical maps from OCT measurements in patients with RPE65-LCA compared with mean normal. Top left: mean normal map of ONL thickness; middle left, ONL thickness map in a 7-year-old patient with RPE65-LCA; top right: patient ONL compared with mean normal ONL (percentage). Two other ONL percentage maps are shown on the right (12 and 22 years of age). Bottom left: ranking by percentage of ONL across this wide region of central retina in 17 patients of different ages (age of each patient listed below each bar). Left-hatched bar on the y-axis: lower limit of normal range.
Topographic maps of ONL thickness as a fraction of mean normal are shown in three representative patients with RPE65-LCA for a wide region of central retina (Fig. 2B). Analyses excluded areas around the optic nerve head and the region of highest cone density (i.e., lowest rod to cone ratio; demarcated by squares with hatching). Exclusion of the fovea and surrounding region would leave areas for analysis that have rod to cone ratios of approximately 14:22 and therefore are closer to the mouse ratio of approximately 30:1. 3739 The process leading to the calculation of percentage of ONL thickness is illustrated for a 7-year-old RPE65-LCA patient. There was considerable reduction in ONL thickness and regional retinal differences in disease severity. The superior and superior–temporal retina had a far greater fraction of normal ONL than did the inferior retina (Fig. 2B, top row). The average of this map is 58% of normal. A 22-year-old patient had a similar topographical pattern and 53% of normal ONL. An even lower fraction of normal ONL was present in a 12-year-old patient, and the pattern showed less superior–inferior retinal asymmetry than that in the other two patients (Fig. 2B, right panels). Seventeen RPE65-LCA patients with the highest fractions of ONL calculated by this method were ranked, and the ages at time of mapping are displayed below each ONL fraction (Fig. 2B, left graph). The relationship to age was not pronounced in this group. A tendency toward reduction of ONL thickness with age in the first three decades of life may be present, but there was no statistical significance in a calculated correlation (r 2 = 0.14; P = 0.076). The largest ONL fraction of approximately 60% of normal that we measured in this series of RPE65-LCA patients would be similar to the amount of ONL in 8- to 10-month-old Rpe65 −/− mice. 
Age-Related Acceleration of Early Activation Phase of Photoresponses in Rpe65−/− Mice
Severe chromophore deprivation in Rpe65 −/− mice 11 results in severe loss of quantum catch within individual rod photoreceptors, 40 explaining the stereotypical ERG photoresponses at younger ages. 16,17 The leading edges of these ERG photoresponses, when normalized by P3max amplitude, showed substantially shallower slopes in the Rpe65 −/− mice compared with those in the Rpe65 +/+ mice, as exemplified by representative 1-month-old animals (Fig. 3A). The P3sens parameter describing the early activation phase was reduced (1.74 vs. 3.30 log scot-cd−1 · s−1 · m2) and the time to reach the a-wave trough was substantially increased (10.3 vs. 5.2 ms). Unexpectedly, with increasing age, the Rpe65 −/− mice showed an acceleration of the leading edge of ERG photoresponses, such that by 16 months of age they had time courses essentially equivalent to those of the aged Rpe65 +/+ mice (Fig. 3A). This acceleration is not an artifact of the amplitude normalization; unscaled responses show initiation of leading edges occurring much earlier in older than in the younger Rpe65 −/− mice (data not shown). The natural history of the P3sens parameter in the Rpe65 +/+ mice can be described with the log-linear function, log(P3sens) = 3.53 − 0.011 · months, which was essentially flat with age (Fig. 3B, left). In Rpe65 −/− mice, however, there was an age-related increase in the P3sens parameter between 1 and 12 months. Between 14 and 25 months, there were limited data available above the noise floor, but the results suggest a flattening of the P3sens in the Rpe65 −/− mice, such that a higher order curve had to be used to describe the natural history (Fig. 3B, right). Photoreceptor function and structure results taken together support the conjecture that a greater amount of chromophore becomes available to surviving Rpe65 −/− rods during the slowly progressive photoreceptor degeneration. 
Figure 3.
 
Photoreceptor sensitivity increased with age in the Rpe65 −/− mice. (A) ERG photoresponses in the Rpe65 +/+ and Rpe65 −/− mice of different ages (m), shown normalized by their saturated amplitudes (P3max = 445, 137, 82, 69, 55, 31, 36, and 138 μV, respectively from left to right), to emphasize differences in the shape of their leading edges. Thin traces: ERGs; thick traces: the phototransduction model fit as an ensemble. All traces start at flash onset; poststimulus time point corresponding to 5 ms shown duplicated in each panel to allow comparison. At 1 month, the Rpe65 −/− photoresponses were slower than the Rpe65 +/+. Between 3 and 16 months, the Rpe65 −/− photoresponses progressively accelerated, such that they were essentially identical in shape to the older Rpe65 +/+ photoresponse. (B) Photoresponse sensitivity (P3sens) parameter in the Rpe65 +/+ and Rpe65 −/− mice as a function of age. With age, there was a slight decline of P3sens in the Rpe65 +/+, whereas there was a dramatic increase in the Rpe65 −/− mice. Log(P3sens) as a function of age can be described (thick lines) with a linear relationship for the Rpe65 +/+mice, but at least a second-order function was necessary for the Rpe65 −/− mice.
Figure 3.
 
Photoreceptor sensitivity increased with age in the Rpe65 −/− mice. (A) ERG photoresponses in the Rpe65 +/+ and Rpe65 −/− mice of different ages (m), shown normalized by their saturated amplitudes (P3max = 445, 137, 82, 69, 55, 31, 36, and 138 μV, respectively from left to right), to emphasize differences in the shape of their leading edges. Thin traces: ERGs; thick traces: the phototransduction model fit as an ensemble. All traces start at flash onset; poststimulus time point corresponding to 5 ms shown duplicated in each panel to allow comparison. At 1 month, the Rpe65 −/− photoresponses were slower than the Rpe65 +/+. Between 3 and 16 months, the Rpe65 −/− photoresponses progressively accelerated, such that they were essentially identical in shape to the older Rpe65 +/+ photoresponse. (B) Photoresponse sensitivity (P3sens) parameter in the Rpe65 +/+ and Rpe65 −/− mice as a function of age. With age, there was a slight decline of P3sens in the Rpe65 +/+, whereas there was a dramatic increase in the Rpe65 −/− mice. Log(P3sens) as a function of age can be described (thick lines) with a linear relationship for the Rpe65 +/+mice, but at least a second-order function was necessary for the Rpe65 −/− mice.
Inner Retinal Changes in Rpe65−/− Mice and Human RPE65-LCA
Dark-adapted ERGs evoked with bright flashes show a cornea-negative P3 component dominated by photoreceptor activity, a cornea-positive P2 component dominated by bipolar cell activity, and transient oscillatory potentials (OPs) that are driven by inner retinal activity. 41,42 Normalization of postreceptoral components by the photoreceptor function can provide insight into understanding changes in signaling to the visual system by remaining photoreceptors upon degeneration of their neighbors. 12,14,43 Dark-adapted ERG photoresponses evoked with 3.62 log scot-cd · s · m−2 stimuli in the younger and older Rpe65 +/+ mice exemplify these components (Fig. 4A, left). When normalized by the photoreceptor amplitude (P3max), the younger and older mice appeared to have identical ERGs. The natural history of the P280/P3max ratio, capturing bipolar cell function normalized by the photoreceptor function (Fig. 3B), was age-invariant (F = 0.34; P = 0.56), even though each parameter declined individually with age (Fig. 1). 
Figure 4.
 
Inner retinal function in the Rpe65 −/− mouse and structure in human RPE65-LCA. (A) Representative ERG waveforms elicited with a 3.6 log scot-cd · s · m−2 flash in (black traces) 3- and (gray traces) 16-month-old Rpe65 +/+ and Rpe65 −/− mice. Right: the same responses are superimposed after equating a-wave amplitude. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (B) Ratio of amplitude dominated by bipolar cell function (P280) and photoreceptor function (P3max) plotted as a function of age in the Rpe65 +/+ (left) and Rpe65 −/− (right) mice. Linear regression (thick line) and 95% prediction intervals (thin lines) are shown. (C) Relationship of outer and inner laminar features in human RPE65-mutant retinas. Inner retinal thickness as a function of ONL thickness at extrafoveal (>2 mm of eccentricity; 0.3 mm bins) retinal locations in the patients and in normal subjects. Both inner retinal and ONL thicknesses are specified as change from mean normal value calculated at each retinal location. Small and large circular symbols, color coded for patients of different ages, are shown to the right of the graph; normal subjects, gray squares. Small circles: individual loci; large circles: the average of all loci studied in the patient; vertical dashed line: the lower normal limit (−2 SD from mean normal) of ONL thickness; horizontal dashed line: the upper normal limit (+2SD from mean normal) of inner retinal thickness.
Figure 4.
 
Inner retinal function in the Rpe65 −/− mouse and structure in human RPE65-LCA. (A) Representative ERG waveforms elicited with a 3.6 log scot-cd · s · m−2 flash in (black traces) 3- and (gray traces) 16-month-old Rpe65 +/+ and Rpe65 −/− mice. Right: the same responses are superimposed after equating a-wave amplitude. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (B) Ratio of amplitude dominated by bipolar cell function (P280) and photoreceptor function (P3max) plotted as a function of age in the Rpe65 +/+ (left) and Rpe65 −/− (right) mice. Linear regression (thick line) and 95% prediction intervals (thin lines) are shown. (C) Relationship of outer and inner laminar features in human RPE65-mutant retinas. Inner retinal thickness as a function of ONL thickness at extrafoveal (>2 mm of eccentricity; 0.3 mm bins) retinal locations in the patients and in normal subjects. Both inner retinal and ONL thicknesses are specified as change from mean normal value calculated at each retinal location. Small and large circular symbols, color coded for patients of different ages, are shown to the right of the graph; normal subjects, gray squares. Small circles: individual loci; large circles: the average of all loci studied in the patient; vertical dashed line: the lower normal limit (−2 SD from mean normal) of ONL thickness; horizontal dashed line: the upper normal limit (+2SD from mean normal) of inner retinal thickness.
The inner retinal function in the Rpe65 −/− mice was different from that in the Rpe65 +/+ mice. The leading edge of the P2 component appeared to rise more slowly in the mutant animals (Fig. 4A, right). In addition, there could be prominent OPs with an amplitude envelope extending to times later than that of the Rpe65 +/+. With increasing age, there was a tendency of the Rpe65 -/− mice to retain a postreceptoral component disproportionate to the reduced receptoral component, as illustrated by the waveforms of the older and younger animals normalized by P3max (Fig. 4A, right). In some older animals, OPs were the only physiological signal remaining in a waveform with otherwise nondetectable photoreceptor and bipolar cell components. The apparent retention of the inner retinal signaling showed variability but the natural history of the P280/P3max ratio showed a linear increase with age with a non-0 slope (slope = 0.08 month−1; F = 11.52; P = 0.001), between the ages of 1 and 16 months (Fig. 4B, right). 
A direct comparison of ERG results in the Rpe65 −/− mouse with those in human RPE65-LCA is not possible, because there are no rod-based ERGs in most RPE65-LCA patients and no detectable waveform with brighter stimuli that evoke photoreceptor responses. 10 Is there any detectable inner retinal disease in RPE65-LCA that can be identified with other noninvasive studies? In 10 RPE65-LCA patients (ages, 12–40), we explored the association between photoreceptor loss and inner retinal thickening, a surrogate for retinal remodeling in many retinal degenerations including other forms of LCA (Fig. 4C) (for example, Refs. 2325, 4446). All the RPE65-LCA patients had loci with ONL thickness that was abnormally reduced to various degrees, but two patients (ages, 12 and 24) also showed some loci that were within the normal range. Of the 1841 loci sampled, most (n = 1802; 97.7%) were abnormally reduced in ONL thickness. The inner retinal measurements indicated that there were no loci with thinning. Inner retinal thickness was either within normal limits (n = 589; 32%) or was hyperthick (n = 1252; 68%). Patients at the extremes of age studied (ages, 12 and 40) showed different patterns of ONL versus inner retinal thickness. The youngest patient (age 12) had a spectrum of ONL changes, from no significant change to only 40% of normal thickness, and all the associated inner retinal measurements were normal. In contrast, the oldest patient (age 40) had the most hyperthick inner retina, and ONL thicknesses were among the lowest. Averages of all loci in each patient (larger symbols superimposed on the individual data) suggest a trend toward increased inner retinal thickening with decreased ONL. 
Discussion
We sought to elucidate the time course of disease expression resulting from Rpe65 deficiency by providing a quantitative description of the ERG abnormalities in Rpe65 −/− mice compared with those in control mice over a wide age range from 1 to 25 months. Considering the prominent retinal degeneration in RPE65-LCA patients of all ages, 8,9,20,47 a natural history study in Rpe65-deficient mice should help define the stages of murine disease that are more relevant to future clinical trials than the earliest stages of murine disease that have been used as proof-of-concept for translation to current human clinical trials. 
Natural History of Photoreceptor Amplitude in Rpe65−/− Mice
Photoreceptor (P3) components of the ERG were quantified with a phototransduction activation model defined by two free parameters: P3max and P3sens. 1214 The maximum-amplitude parameter of the photoreceptor component (P3max) is believed to be proportional to the total number of cyclic nucleotide-gated channels open on rod outer segment (ROS) plasma membranes in the dark across the retina. 48,49 At the youngest ages (1–3 months), P3max of Rpe65 −/− was ∼25% of Rpe65 +/+, consistent with a previous report. 16 Reduction of P3max is commonly related to degenerative changes involving a loss of ROS plasma membrane area secondary to OS shortening and rod loss, 12,5052 but the structure of Rpe65 −/− photoreceptors has been reported to be near normal at these ages. 11,33,34,36 P3max reduction can also be due to nondegenerative changes in the circulating dark current. Single cell recordings from Rpe65 −/− rods show a substantial drop in the dark current to ∼10%–16% of control, 32,35,40,53 which is hypothesized to result from spontaneous low-level activation of copious amounts of free opsin found in these chromophore-deprived but structurally intact retinas 32,25 modified by constitutive phosphorylation. 30,53 Supporting a nondegenerative basis of the P3max reduction measured in young Rpe65-deficient retinas is our previous observation of a substantial increase in saturated amplitude within 48 hours of oral treatment with a substitute chromophore. 16  
P3max in Rpe65 −/− mice showed a progressive reduction over the age range from 1 to 25 months; an exponential function could reasonably approximate the progressive reduction with a time constant of ∼11 months. The rate of reduction appeared to track closely the natural history of Rpe65 −/− photoreceptor loss, as estimated from ONL thicknesses measured or illustrated in previous publications. These results suggest that independent of the specific cause of the P3max reduction in the young mice, the ensuing age-related decline was probably caused by the loss of photoreceptors. 
Natural History of the Early Activation Phase of Photoresponses in Rpe65−/− Mice
The sensitivity parameter of the ERG photoresponse (P3sens) provides a quantitative in vivo measure of phototransduction amplification gain averaged across all functioning photoreceptors of the retina. 12,14,48 P3sens was significantly reduced in Rpe65 −/− at 1 month of age, consistent with previous results. 16,17 Qualitatively, a reduction in P3sens would be consistent with the expected loss of quantum catch in Rpe65 −/− photoreceptors starved of chromophore. 11 Quantitatively, the 1.3-log-unit P3sens reduction measured in vivo was smaller than the 2- to 3-log-unit change in peak sensitivity (reciprocal of half-saturating flash intensity) measured from Rpe65 −/− rods in isolated retinas. 35,40,53 It is important to note that P3sens and peak sensitivity are not directly comparable: the first measure is dominated by early (<20 ms) activation reactions, whereas the second measure includes the effects of activation as well as deactivation reactions at later (>100 ms) times during phototransduction. 54 Interpretation of the apparent differences in measures of photoreceptor responsiveness must await ERG and single-cell recordings performed in the same eyes controlling for the small amounts of the remnant chromophore driving these visual responses. 
Unexpected was the finding of a progressively accelerating early activation phase of ERG photoresponses (P3sens) with age in Rpe65 −/− mice, such that near-normality was reached by 12 to 15 months age. Several hypotheses, alone or in concert, can be entertained to help explain this finding. First, it may be argued that with greater photoreceptor degeneration, there is less ROS volume and thus less opsin molecules available with age. Assuming that the rate of remnant chromophore production in Rpe65 −/− remains invariant with age, there could be an effective reduction in the number of free opsin molecules. Such an effect was hypothesized to occur when Rpe65 −/− degeneration accelerated with elimination of phosphorylation. 53 One predicted result of the reduction of free opsin concentration would be an increase in the dark current, and consequent increase in P3max. The fact that P3max is tracking the retinal degeneration, however, implies that the intrinsic dark current does not change substantially with age, beyond that explained by shorter ROS. A second possibility involves a greater contribution of cone photoreceptor signaling with aging within the leading edge of the ERG photoresponse. In wild-type mice, a small, cone-derived component can indeed be demonstrated. 55 However, substantial numbers of cones in Rpe65 −/− mice are lost early in the disease 5659 making the cone hypothesis unlikely. A third hypothesis involves an increase in regenerated opsin concentration with age. The source of the chromophore that regenerates opsin in Rpe65 −/− rods remains unknown, 31,40 but its significance in driving relatively large ERG waveforms in Rpe65 −/− mice is remarkable. If retinyl esters were the substrate for the unknown isomerization reaction producing this biochemically tiny amount of chromophore, then the well-established accumulation of retinyl esters with age in Rpe65 −/− mice 11,16,32,36,6062 may be relevant. Of note, an age-related increase in retinaldehydes in Rpe65 −/− retinas has been reported, although the amounts were near detectability limits of the methods used. 61 A test of this hypothesis may involve the natural history of Lrat −/− mice which, at young ages, also show loss of P3sens and a severe chromophore deficiency but, unlike Rpe65 −/− mice, do not accumulate retinyl esters. 63  
Comparison of the Results with the Human Disease They Are Intended to Model
Like the patients with RPE65-LCA, there was progressive loss of visual function in the Rpe65 −/− mice. Visual function in RPE65-LCA has been quantified by using focal and full-field psychophysical techniques and full-field ERGs in groups of patients with different RPE65 genotypes. 10,20,64 Rod visual function is detectable, albeit severely reduced, in some patients but only rarely after the third decade of life. 10 Cone function measured by visual acuity or with other psychophysical methods can persist for decades. 10,20 Kinetic visual field extent varies widely in the first two decades of life and becomes extremely limited by the third decade. 10,64 ERG techniques similar to those used in mice have shown that patients have nondetectable ERGs or very severe losses of retinal function, even in the first decade of life. Unlike the patients, retinal function is measurable over almost the entire lifespan of the animal. Our estimates of ONL loss in a wide expanse of central retina in RPE65-LCA patients ages 6 to 17 indicate that, on average, there is at least a 50% loss. 9 In the present study, the highest ONL fraction compared with normal among relatively young patients with RPE65-LCA was 58%. Such estimates in humans would relate to near the end of the first year of life in the Rpe65 −/− mouse. Ideally, experiments of efficacy of any treatment in this mouse would be performed at this age. To be considered in this recommendation is the fact that, at 9 months of age, there is still some deficit in the photoresponse activation phase, and correction may be easier to demonstrate; older mice have a near-normal activation phase and it may be more difficult to show a treatment effect at the level of photoreceptors. 
Inner Retinal Abnormalities in the Rpe65−/− Mouse and in RPE65-LCA
A topic of importance in treating all disease stages is the state of inner retinal function, and this aspect was also explored in the present study. With the human visual data that can be collected in RPE65-LCA patients, there is no way to determine whether there is any inner retinal dysfunction that contributes to the profound visual disturbance. In fact, the relative preservation of ONL compared with visual loss, when quantified, is not unlike the pattern in congenital stationary night blindness, although the latter has little or no retinal degenerative component. 65 The question about inner retinal function thus becomes an important one to ask in the mouse models with recordable signals. 
With age in the Rpe65 −/− mice, there was a trend toward inner retinal abnormality, as measured by the P2 to P3 ratio. The relatively large variation in this ratio in the mutant animals versus the Rpe65 +/+ mice may be related to other inherited features of the inner retina independent of the Rpe65 deficiency. There is the possibility that the effect is similar to the relatively augmented rod bipolar cell function that has been described in another rodent model of hereditary retinal degeneration, the P23H rhodopsin transgenic rat model of autosomal dominant retinitis pigmentosa. 12 The present study is the first to provide data on retinal remodeling in human RPE65-LCA, such as has been published for other retinal degenerations (for example, Refs. 24,25,4446). The trend toward thickening of the inner retina with increasing loss of photoreceptors in the RPE65-LCA patients is consistent with a similar quantitative analysis in patients with XLRP caused by mutations in the RPGR gene. 25 Unlike the results in our study of RPGR-XLRP (which also included young patients), two of the RPE65-LCA patients had loss of photoreceptors but little or no thickening of the inner retina. Also, the thickening was more pronounced in RPGR-XLRP, with some inner retinal thickening measuring about twice that of the most extreme thickening in RPE65-LCA. The basis for this difference is unknown. 
Clinical Implications of the Comparison of Murine and Human Diseases
What is in the future for gene therapy of RPE65-LCA? A possible scenario once current versions of the vector-gene product are marketed is that LCA patients and their families will seek molecular genetic diagnosis after a clinical diagnosis is made and, if disease-causing mutations in RPE65 are found, there will be requests for the available treatment (independent of age and disease stage). After subretinal injection, some patients will have noticeable increases in vision and others will not. The basis for limited efficacy or complete failure will not be specifically known but, in an otherwise incurable condition, it is likely to be accepted and excused as simply a gamble worth taking. Parallel to the availability of this first product, there may also be progress in other treatment strategies of the same disorder, such as with more efficient or specific viral vectors, 6669 nonviral nanoparticle methods, 70 transretinal delivery by vitreal injection, 71 or the evolution of oral 9-cis retinoids as a complement or substitute for a gene-based therapy. 16,72  
The relatively rapid translation from preclinical studies to human trials for RPE65-LCA has left many unanswered questions about the indications of treatment and subsequent effects, once treatment is delivered. Whereas most studies of young Rpe65-deficient animals were exceedingly valuable to begin the clinical trial work, they are now less helpful in the decision on how to continue and enhance it. Only rare reports have inquired about efficacy in later-stage Rpe65-deficient mice with retinal degeneration more akin to all ages in humans. 8,73 The present studies suggest that further experiments would be worthwhile to perform in Rpe65 −/− mice toward the end of the first year of life, inquiring about outcomes at this disease stage, determining the state of inner retinal anatomic as well as physiological abnormalities and their impact on outcome, and answering the key question of longevity of the treatment effect. Questions about longevity of treated retina in the presence of degenerating retina are far more feasible to answer over a few months in the lifespan of the Rpe65 −/− mouse than over many years in patients with RPE65-LCA. Given further experiments, there may be hypotheses to test and opportunities to enhance treatment outcome in the mice rather than simply treating every molecularly positive LCA patient, assuming that detachment of more retina is better than less with subretinal injection(s), advocating for decreases in age at which such treatment can be administered, and hoping for better and persistent efficacy but not being able to predict what leads to it. 
Footnotes
 Supported by grants from the Macula Vision Research Foundation, Hope for Vision, the Chatlos Foundation, and the Foundation Fighting Blindness.
Footnotes
 Disclosure: R.C. Caruso, None; T.S. Aleman, None; A.V. Cideciyan, None; A.J. Roman, None; A. Sumaroka, None; C.L. Mullins, None; S.L. Boye, None; W.W. Hauswirth, None; S.G. Jacobson, None
References
Stone EM . Leber congenital amaurosis-a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture. Am J Ophthalmol. 2007;144:791–811. [CrossRef] [PubMed]
den Hollander AI Roepman R Koenekoop RK Cremers FP . Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res. 2008;27:391–419. [CrossRef] [PubMed]
Cideciyan AV . Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Prog Retin Eye Res. 2010;29:398–427. [CrossRef] [PubMed]
Bainbridge JW Smith AJ Barker SS . Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med. 2008;358:2231–2239. [CrossRef] [PubMed]
Maguire AM Simonelli F Pierce EA . Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 2008;358:2240–2248. [CrossRef] [PubMed]
Hauswirth WW Aleman TS Kaushal S . Treatment of Leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short term results of a phase I trial. Hum Gene Ther. 2008;19:979–990. [CrossRef] [PubMed]
Cideciyan AV Aleman TS Boye SL . Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci USA. 2008;105:15112–15117. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV . Identifying photoreceptors in blind eyes caused by RPE65 mutations: prerequisite for human gene therapy success. Proc Natl Acad Sci USA. 2005;102:6177–6182. [CrossRef] [PubMed]
Jacobson SG Cideciyan AV Aleman TS . Photoreceptor layer topography in children with Leber congenital amaurosis caused by RPE65 mutations. Invest Ophthalmol Vis Sci. 2008;49:4573–4577. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV . Defining the residual vision in Leber congenital amaurosis caused by RPE65 mutations. Invest Ophthalmol Vis Sci. 2009;50:2368–2375. [CrossRef] [PubMed]
Redmond TM Yu S Lee E . Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998;20:344–351. [CrossRef] [PubMed]
Aleman TS LaVail MM Montemayor R . Augmented rod bipolar cell function in partial receptor loss: an ERG study in P23H rhodopsin transgenic and aging normal rats. Vision Res. 2001;41:2779–2797. [CrossRef] [PubMed]
Roman AJ Boye SL Aleman TS . Electroretinographic analyses of Rpe65-mutant rd12 mice: developing an in vivo bioassay for human gene therapy trials of Leber congenital amaurosis. Mol Vis. 2007;13:1701–1710. [PubMed]
Cideciyan AV Jacobson SG . An alternative phototransduction model for human rod and cone ERG a-waves: normal parameters and variation with age. Vision Res. 1996;36:2609–2621. [CrossRef] [PubMed]
Cideciyan AV . In vivo assessment of photoreceptor function in human diseases caused by photoreceptor-specific gene mutations. Methods Enzymol. 2000;316:611–626. [PubMed]
Van Hooser JP Aleman TS He YG . Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness. Proc Natl Acad Sci USA. 2000;97:8623–8628. [CrossRef] [PubMed]
Dejneka NS Surace EM Aleman TS . In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther. 2004;9:182–188. [CrossRef] [PubMed]
Lamb TD Pugh ENJr . Phototransduction, dark adaptation, and rhodopsin regeneration: the Proctor Lecture. Invest Ophthalmol Vis Sci. 2006;47:5137–5152. [CrossRef] [PubMed]
Hood DC Birch DG . A computational model of the amplitude and implicit time of the b-wave of the human ERG. Vis Neurosci. 1992;8:107–126. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV . Human cone photoreceptor dependence on RPE65 isomerase. Proc Natl Acad Sci USA. 2007;104:15123–15128. [CrossRef] [PubMed]
Huang Y Cideciyan AV Papastergiou GI . Relation of optical coherence tomography to microanatomy in normal and rd chickens. Invest Ophthalmol Vis Sci. 1998;39:2405–2416. [PubMed]
Jacobson SG Cideciyan AV Aleman TS . Crumbs homolog 1 (CRB1) mutations result in a thick human retina with abnormal lamination. Hum Mol Genet. 2003;12:1073–1078. [CrossRef] [PubMed]
Aleman TS Cideciyan AV Sumaroka A . Retinal laminar architecture in human retinitis pigmentosa caused by Rhodopsin gene mutations. Invest Ophthalmol Vis Sci. 2008;49:1580–1590. [CrossRef] [PubMed]
Jacobson SG Cideciyan AV Sumaroka A . Remodeling of the human retina in choroideremia: rab escort protein 1 (REP-1) mutations. Invest Ophthalmol Vis Sci. 2006;47:4113–4120. [CrossRef] [PubMed]
Aleman TS Cideciyan AV Sumaroka A . Inner retinal abnormalities in X-linked retinitis pigmentosa with RPGR mutations. Invest Ophthalmol Vis Sci. 2007;48:4759–4765. [CrossRef] [PubMed]
Drasdo N Millican CL Katholi CR Curcio CA . The length of Henle fibers in the human retina and a model of ganglion receptive field density in the visual field. Vision Res. 2007;47:2901–2911. [CrossRef] [PubMed]
Li C Cheng M Yang H Peachey NS Naash MI . Age-related changes in the mouse outer retina. Optom Vis Sci. 2001;78:425–430. [CrossRef] [PubMed]
Gresh J Goletz PW Crouch RK Rohrer B . Structure-function analysis of rods and cones in juvenile, adult, and aged C57bl/6 and Balb/c. Vis Neurosci. 2003;20:211–220. [CrossRef] [PubMed]
Williams GA Jacobs GH . Cone-based vision in the aging mouse. Vision Res. 2007;47:2037–2046. [CrossRef] [PubMed]
Ablonczy Z Crouch RK Goletz PW . 11-cis-Retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors. J Biol Chem. 2002;277:40491–40498. [CrossRef] [PubMed]
Fan J Rohrer B Moiseyev G Ma JX Crouch RK . Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice. Proc Natl Acad Sci USA. 2003;100:13662–13667. [CrossRef] [PubMed]
Woodruff ML Wang Z Chung HY Redmond TM Fain GL Lem J . Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis. Nat Genet. 2003;35:158–164. [CrossRef] [PubMed]
Gouras P Kong J Tsang SH . Retinal degeneration and RPE transplantation in Rpe65(−/−) mice. Invest Ophthalmol Vis Sci. 2002;43:3307–3311. [PubMed]
Rohrer B Goletz P Znoiko S . Correlation of regenerable opsin with rod ERG signal in Rpe65−/− mice during development and aging. Invest Ophthalmol Vis Sci. 2003;44:310–315. [CrossRef] [PubMed]
Fan J Woodruff ML Cilluffo MC Crouch RK Fain GL . Opsin activation of transduction in the rods of dark-reared Rpe65 knockout mice. J Physiol. 2005;568:83–95. [CrossRef] [PubMed]
Samardzija M von Lintig J Tanimoto N . R91W mutation in Rpe65 leads to milder early onset retinal dystrophy due to the generation of low levels of 11-cis-retinal. Hum Mol Genet. 2008;17:281–292. [CrossRef] [PubMed]
Curcio CA Sloan KR Kalina RE Hendrickson AE . Human photoreceptor topography. J Comp Neurol. 1990;292:497–523. [CrossRef] [PubMed]
Carter-Dawson LD LaVail MM . Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy. J Comp Neurol. 1979;188(2):245–262. [CrossRef] [PubMed]
Jeon C-J Strettoi E Masland RH . The major cell populations of the mouse retina. J Neurosci. 1998;18:8936–8946. [PubMed]
Van Hooser JP Liang Y Maeda T . Recovery of visual functions in a mouse model of Leber congenital amaurosis. J Biol Chem. 2002;277:19173–19182. [CrossRef] [PubMed]
Wachtmeister L . Oscillatory potentials in the retina: what do they reveal. Prog Retin Eye Res. 1998;17:485–521. [CrossRef] [PubMed]
Weymouth AE Vingrys AJ . Rodent electroretinography: methods for extraction and interpretation of rod and cone responses. Prog Retin Eye Res. 2008;27:1–44. [CrossRef] [PubMed]
Banin E Cideciyan AV Aleman TS . Retinal rod photoreceptor-specific gene mutation perturbs cone pathway development. Neuron. 1999;23:549–557. [CrossRef] [PubMed]
Jacobson SG Sumaroka A Aleman TS Cideciyan AV Danciger M Farber DB . Evidence for retinal remodeling in retinitis pigmentosa caused by PDE6B mutation. Br J Ophthalmol. 2007;91:699–701. [CrossRef] [PubMed]
Cideciyan AV Aleman TS Jacobson SG . Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis. Hum Mut. 2007;28:1074–1083. [CrossRef] [PubMed]
Jacobson SG Aleman TS Cideciyan AV . Leber congenital amaurosis caused by Lebercilin (LCA5) mutation: retained photoreceptors adjacent to retinal disorganization. Mol Vis. 2009;15:1098–1106. [PubMed]
Maeda T Maeda A Casadesus G Palczewski K Margaron P . Evaluation of 9-cis-retinyl acetate therapy in Rpe65−/− mice. Invest Ophthalmol Vis Sci. 2009;50:4368–4378. [CrossRef] [PubMed]
Pugh ENJr Lamb TD . Amplification and kinetics of the activation steps in phototransduction. Biochim Biophys Acta. 1993;1141:111–149. [CrossRef] [PubMed]
Pugh ENJr Falsini B Lyubarsky AL . The origin of the major rod- and cone-driven components of the rodent electroretinogram, and the effect of age and light-rearing history on the magnitudes of these components. In: Williams TP Thistle AB eds. Photostasis and Related Topics. New York: Plenum; 1998;93–128.
Cideciyan AV Jacobson SG . Negative electroretinograms in retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1993;34:3253–3263. [PubMed]
Hood DC Birch DG . Rod phototransduction in retinitis pigmentosa: estimation and interpretation of parameters derived from the rod a-wave. Invest Ophthalmol Vis Sci. 1994;35:2948–2961. [PubMed]
Goto Y Peachey NS Ziroli NE . Rod phototransduction in transgenic mice expressing a mutant opsin gene. J Opt Soc Am A Opt Image Sci Vis. 1996;13:577–585. [CrossRef] [PubMed]
Fan J Sakurai K Chen CK . Deletion of GRK1 causes retina degeneration through a transducin-independent mechanism. J Neurosci. 2010;30:2496–2503. [CrossRef] [PubMed]
Leskov IB Klenchin VA Handy JW . The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements. Neuron. 2000;27:525–537. [CrossRef] [PubMed]
Lyubarsky AL Falsini B Pennesi ME Valentini P Pugh ENJr . UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments. J Neurosci. 1999;19:442–455. [PubMed]
Znoiko SL Rohrer B Lu K Lohr HR Crouch RK Ma JX . Downregulation of cone-specific gene expression and degeneration of cone photoreceptors in the Rpe65−/− mouse at early ages. Invest Ophthalmol Vis Sci. 2005;46:1473–1479. [CrossRef] [PubMed]
Rohrer B Lohr HR Humphries P Redmond TM Seeliger MW Crouch RK . Cone opsin mislocalization in Rpe65−/− mice: a defect that can be corrected by 11-cis retinal. Invest Ophthalmol Vis Sci. 2005;46:3876–3882. [CrossRef] [PubMed]
Zhang H Fan J Li S . Trafficking of membrane-associated proteins to cone photoreceptor outer segments requires the chromophore 11-cis-retinal. J Neurosci. 2008;28:4008–4014. [CrossRef] [PubMed]
Fan J Rohrer B Frederick JM Baehr W Crouch RK . Rpe65−/− and Lrat−/− mice: comparable models of Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2008;49:2384–2389. [CrossRef] [PubMed]
Moiseyev G Crouch RK Goletz P Oatis JJr. Redmond TM Ma JX . Retinyl esters are the substrate for isomerohydrolase. Biochemistry. 2003;42:2229–2238. [CrossRef] [PubMed]
Qtaishat NM Redmond TM Pepperberg DR . Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice. Invest Ophthalmol Vis Sci. 2003;44:1435–1446. [CrossRef] [PubMed]
Maeda A Maeda T Imanishi Y Golczak M Moise AR Palczewski K . Aberrant metabolites in mouse models of congenital blinding diseases: formation and storage of retinyl esters. Biochemistry. 2006;45:4210–4219. [CrossRef] [PubMed]
Batten ML Imanishi Y Maeda T . Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J Biol Chem. 2004;279:10422–10432. [CrossRef] [PubMed]
Paunescu K Wabbels B Preising MN Lorenz B . Longitudinal and cross-sectional study of patients with early onset severe retinal dystrophy associated with RPE65 mutations. Graefes Arch Clin Exp Ophthalmol. 2005;243:417–426. [CrossRef] [PubMed]
Jacobson SG Cideciyan AV Aleman TS . Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism. Hum Mol Genet. 2008;17:2405–2415. [CrossRef] [PubMed]
Dinculescu A Glushakova L Min SH Hauswirth WW . Adeno-associated virus-vectored gene therapy for retinal disease. Hum Gene Ther. 2005;16:649–663. [CrossRef] [PubMed]
Yáñez-Muñoz RJ Balaggan KS MacNeil A . Effective gene therapy with nonintegrating lentiviral vectors. Nat Med. 2006;12:348–353. [CrossRef] [PubMed]
Lebherz C Maguire A Tang W Bennett J Wilson JM . Novel AAV serotypes for improved ocular gene transfer. J Gene Med. 2008;10:375–382. [CrossRef] [PubMed]
Petrs-Silva H Dinculescu A Li Q . High efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Ther. 2008;17:463–471. [CrossRef] [PubMed]
Farjo R Skaggs J Quiambao AB Cooper MJ Naash MI . Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS One. 2006;1:e38. [CrossRef] [PubMed]
Dalkara D Kolstad KD Caporale N . Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Mol Ther. 2009;17:2096–2102. [CrossRef] [PubMed]
Rotenstreich Y Harats D Shaish A Pras E Michael B . Treatment of a retinal dystrophy, fundus albipunctatus, with oral 9-cis beta-carotene. Br J Ophthalmol. 2010;94(5):616–621. [CrossRef] [PubMed]
Nusinowitz S Ridder WH3rd Pang JJ . Cortical visual function in the rd12 mouse model of Leber congenital amaurosis (LCA) after gene therapy replacement therapy to restore retinal function. Vision Res. 2006;46:3926–3934. [CrossRef] [PubMed]
Figure 1.
 
Retinal function change with age in the Rpe65 +/+ and the Rpe65 −/− mice. (A) ERG luminance-response series obtained in dark-adapted conditions over a range from −4.2 to 3.6 log scot-cd · s · m−2 flashes show elevation of b-wave thresholds in the Rpe65 −/− mice by ∼3 log units compared with those in the Rpe65 +/+ mice at the ages of 3 and 9 months (m). ERG amplitude responses to the highest intensities of light were reduced in the older mouse compared with those in the 3-month-old. All traces start at flash onset. (B) Leading edges of dark-adapted ERG photoresponses evoked with 2.2- and 3.6-log-scot-cd · s · m−2 flashes (thin traces) fit as an ensemble with a model of rod phototransduction activation (thick traces). Representative results are shown for two ages. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (C) Rod photoreceptor function as a function of age estimated with the P3max parameter over the ages from 1 to 25 months in the Rpe65 +/+ (left) and the Rpe65 −/− (right) mice. Regression lines (thick gray) describe log-linear change in the parameters with age; 95% prediction intervals (thin gray lines) encompassing the data are also shown. For Rpe65 −/− mice, only data between 1 and 16 months of age were used for regression analyses, to avoid the uncertainty resulting from near-noise-level responses at ages older than 20 months. (D) Bipolar cell function as a function of age estimated with the P280 parameter with analyses comparable to those in (C).
Figure 1.
 
Retinal function change with age in the Rpe65 +/+ and the Rpe65 −/− mice. (A) ERG luminance-response series obtained in dark-adapted conditions over a range from −4.2 to 3.6 log scot-cd · s · m−2 flashes show elevation of b-wave thresholds in the Rpe65 −/− mice by ∼3 log units compared with those in the Rpe65 +/+ mice at the ages of 3 and 9 months (m). ERG amplitude responses to the highest intensities of light were reduced in the older mouse compared with those in the 3-month-old. All traces start at flash onset. (B) Leading edges of dark-adapted ERG photoresponses evoked with 2.2- and 3.6-log-scot-cd · s · m−2 flashes (thin traces) fit as an ensemble with a model of rod phototransduction activation (thick traces). Representative results are shown for two ages. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (C) Rod photoreceptor function as a function of age estimated with the P3max parameter over the ages from 1 to 25 months in the Rpe65 +/+ (left) and the Rpe65 −/− (right) mice. Regression lines (thick gray) describe log-linear change in the parameters with age; 95% prediction intervals (thin gray lines) encompassing the data are also shown. For Rpe65 −/− mice, only data between 1 and 16 months of age were used for regression analyses, to avoid the uncertainty resulting from near-noise-level responses at ages older than 20 months. (D) Bipolar cell function as a function of age estimated with the P280 parameter with analyses comparable to those in (C).
Figure 2.
 
Photoreceptor nuclear layer thickness loss in Rpe65−/− mice and in humans with RPE65-LCA. (A) Comparison of relative loss of photoreceptor ONL thickness with age to the relative loss of photoreceptor function with age. ONL thicknesses in the Rpe65−/− mice are taken from the literature: (Image not available) Redmond et al.11; (■) Gouras et al.33; (▴) Woodruff et al.32; (▲) Rohrer et al.34; ([Image not available]) Jacobson et al.8; (▾) Fan et al.35; and (♦) Samardzija et al.36 The data were normalized by the thickness of Rpe65+/+ at matched ages, when available. P3max parameter (unfilled symbols) for individual Rpe65−/− mice were divided by the log-linear estimate of Rpe65+/+ natural history at each age. Thick lines: log-linear progression estimates for the relative ONL loss and relative P3max loss. (B) Topographical maps from OCT measurements in patients with RPE65-LCA compared with mean normal. Top left: mean normal map of ONL thickness; middle left, ONL thickness map in a 7-year-old patient with RPE65-LCA; top right: patient ONL compared with mean normal ONL (percentage). Two other ONL percentage maps are shown on the right (12 and 22 years of age). Bottom left: ranking by percentage of ONL across this wide region of central retina in 17 patients of different ages (age of each patient listed below each bar). Left-hatched bar on the y-axis: lower limit of normal range.
Figure 2.
 
Photoreceptor nuclear layer thickness loss in Rpe65−/− mice and in humans with RPE65-LCA. (A) Comparison of relative loss of photoreceptor ONL thickness with age to the relative loss of photoreceptor function with age. ONL thicknesses in the Rpe65−/− mice are taken from the literature: (Image not available) Redmond et al.11; (■) Gouras et al.33; (▴) Woodruff et al.32; (▲) Rohrer et al.34; ([Image not available]) Jacobson et al.8; (▾) Fan et al.35; and (♦) Samardzija et al.36 The data were normalized by the thickness of Rpe65+/+ at matched ages, when available. P3max parameter (unfilled symbols) for individual Rpe65−/− mice were divided by the log-linear estimate of Rpe65+/+ natural history at each age. Thick lines: log-linear progression estimates for the relative ONL loss and relative P3max loss. (B) Topographical maps from OCT measurements in patients with RPE65-LCA compared with mean normal. Top left: mean normal map of ONL thickness; middle left, ONL thickness map in a 7-year-old patient with RPE65-LCA; top right: patient ONL compared with mean normal ONL (percentage). Two other ONL percentage maps are shown on the right (12 and 22 years of age). Bottom left: ranking by percentage of ONL across this wide region of central retina in 17 patients of different ages (age of each patient listed below each bar). Left-hatched bar on the y-axis: lower limit of normal range.
Figure 3.
 
Photoreceptor sensitivity increased with age in the Rpe65 −/− mice. (A) ERG photoresponses in the Rpe65 +/+ and Rpe65 −/− mice of different ages (m), shown normalized by their saturated amplitudes (P3max = 445, 137, 82, 69, 55, 31, 36, and 138 μV, respectively from left to right), to emphasize differences in the shape of their leading edges. Thin traces: ERGs; thick traces: the phototransduction model fit as an ensemble. All traces start at flash onset; poststimulus time point corresponding to 5 ms shown duplicated in each panel to allow comparison. At 1 month, the Rpe65 −/− photoresponses were slower than the Rpe65 +/+. Between 3 and 16 months, the Rpe65 −/− photoresponses progressively accelerated, such that they were essentially identical in shape to the older Rpe65 +/+ photoresponse. (B) Photoresponse sensitivity (P3sens) parameter in the Rpe65 +/+ and Rpe65 −/− mice as a function of age. With age, there was a slight decline of P3sens in the Rpe65 +/+, whereas there was a dramatic increase in the Rpe65 −/− mice. Log(P3sens) as a function of age can be described (thick lines) with a linear relationship for the Rpe65 +/+mice, but at least a second-order function was necessary for the Rpe65 −/− mice.
Figure 3.
 
Photoreceptor sensitivity increased with age in the Rpe65 −/− mice. (A) ERG photoresponses in the Rpe65 +/+ and Rpe65 −/− mice of different ages (m), shown normalized by their saturated amplitudes (P3max = 445, 137, 82, 69, 55, 31, 36, and 138 μV, respectively from left to right), to emphasize differences in the shape of their leading edges. Thin traces: ERGs; thick traces: the phototransduction model fit as an ensemble. All traces start at flash onset; poststimulus time point corresponding to 5 ms shown duplicated in each panel to allow comparison. At 1 month, the Rpe65 −/− photoresponses were slower than the Rpe65 +/+. Between 3 and 16 months, the Rpe65 −/− photoresponses progressively accelerated, such that they were essentially identical in shape to the older Rpe65 +/+ photoresponse. (B) Photoresponse sensitivity (P3sens) parameter in the Rpe65 +/+ and Rpe65 −/− mice as a function of age. With age, there was a slight decline of P3sens in the Rpe65 +/+, whereas there was a dramatic increase in the Rpe65 −/− mice. Log(P3sens) as a function of age can be described (thick lines) with a linear relationship for the Rpe65 +/+mice, but at least a second-order function was necessary for the Rpe65 −/− mice.
Figure 4.
 
Inner retinal function in the Rpe65 −/− mouse and structure in human RPE65-LCA. (A) Representative ERG waveforms elicited with a 3.6 log scot-cd · s · m−2 flash in (black traces) 3- and (gray traces) 16-month-old Rpe65 +/+ and Rpe65 −/− mice. Right: the same responses are superimposed after equating a-wave amplitude. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (B) Ratio of amplitude dominated by bipolar cell function (P280) and photoreceptor function (P3max) plotted as a function of age in the Rpe65 +/+ (left) and Rpe65 −/− (right) mice. Linear regression (thick line) and 95% prediction intervals (thin lines) are shown. (C) Relationship of outer and inner laminar features in human RPE65-mutant retinas. Inner retinal thickness as a function of ONL thickness at extrafoveal (>2 mm of eccentricity; 0.3 mm bins) retinal locations in the patients and in normal subjects. Both inner retinal and ONL thicknesses are specified as change from mean normal value calculated at each retinal location. Small and large circular symbols, color coded for patients of different ages, are shown to the right of the graph; normal subjects, gray squares. Small circles: individual loci; large circles: the average of all loci studied in the patient; vertical dashed line: the lower normal limit (−2 SD from mean normal) of ONL thickness; horizontal dashed line: the upper normal limit (+2SD from mean normal) of inner retinal thickness.
Figure 4.
 
Inner retinal function in the Rpe65 −/− mouse and structure in human RPE65-LCA. (A) Representative ERG waveforms elicited with a 3.6 log scot-cd · s · m−2 flash in (black traces) 3- and (gray traces) 16-month-old Rpe65 +/+ and Rpe65 −/− mice. Right: the same responses are superimposed after equating a-wave amplitude. Note differences in calibration bars and substantially smaller responses from the Rpe65 −/− mice. (B) Ratio of amplitude dominated by bipolar cell function (P280) and photoreceptor function (P3max) plotted as a function of age in the Rpe65 +/+ (left) and Rpe65 −/− (right) mice. Linear regression (thick line) and 95% prediction intervals (thin lines) are shown. (C) Relationship of outer and inner laminar features in human RPE65-mutant retinas. Inner retinal thickness as a function of ONL thickness at extrafoveal (>2 mm of eccentricity; 0.3 mm bins) retinal locations in the patients and in normal subjects. Both inner retinal and ONL thicknesses are specified as change from mean normal value calculated at each retinal location. Small and large circular symbols, color coded for patients of different ages, are shown to the right of the graph; normal subjects, gray squares. Small circles: individual loci; large circles: the average of all loci studied in the patient; vertical dashed line: the lower normal limit (−2 SD from mean normal) of ONL thickness; horizontal dashed line: the upper normal limit (+2SD from mean normal) of inner retinal thickness.
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