Moderate Light-Induced Degeneration of Rod Photoreceptors with Delayed Transducin Translocation in shaker1 Mice

Abstract

Purpose.: Usher syndrome is characterized by congenital deafness associated with retinitis pigmentosa (RP). Mutations in the myosin VIIa gene (MYO7A) cause a common and severe subtype of Usher syndrome (USH1B). Shaker1 mice have mutant MYO7A. They are deaf and have vestibular dysfunction but do not develop photoreceptor degeneration. The goal of this study was to investigate abnormalities of photoreceptors in shaker1 mice.

Methods.: Immunocytochemistry and hydroethidine-based detection of intracellular superoxide production were used. Photoreceptor cell densities under various conditions of light/dark exposures were evaluated.

Results.: In shaker1 mice, the rod transducin translocation is delayed because of a shift of its light activation threshold to a higher level. Even moderate light exposure can induce oxidative damage and significant rod degeneration in shaker1 mice. Shaker1 mice reared under a moderate light/dark cycle develop severe retinal degeneration in less than 6 months.

Conclusions.: These findings show that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation. Importantly, USH1B animal models are likely vulnerable to light-induced photoreceptor damage, even under moderate light.

Usher syndrome is the leading genetic disorder resulting in combined blindness and deafness. The main clinical symptoms of the disease are retinitis pigmentosa (RP) and hearing loss. Affected individuals have a sensorineural hearing impairment at birth and later develop progressive RP. ¹ Vestibular dysfunction ² and sometimes mental disturbances ³ are also features of the syndrome. Considering the tremendous burden imposed by the loss of both major senses, it is important to pursue research into the causes of Usher syndrome in the hope that someday an effective therapy may be possible. Usher syndrome is clinically and genetically heterogeneous, and can be divided into three major types (I, II, and III) according to clinical observations distinguished by severity and progression of hearing loss along with the presence or absence of vestibular dysfunction. They can be further divided into different genetic subtypes. Different genes associated with the various subtypes of Usher syndrome have been identified, ^{4 –7} but the mechanism leading to RP in Usher syndrome remains unknown. 

Mutations in the myosin VIIa gene (MYO7A) cause a major subtype of Usher syndrome, type 1B (USH1B), ⁸ , which is characterized by severe deafness at birth and early onset of RP that leads to blindness. MYO7A encodes an unconventional myosin that is present in a variety of tissues. In the retina of various vertebrates, MYO7A protein is expressed in the retinal pigment epithelium (RPE), where it appears to function in maintaining the appropriate distribution of melanosomes. ^9,10 MYO7A is also detected in the photoreceptor cells at cilium region and synaptic terminals. Shaker1 mice have mutations in MYO7A and are widely accepted animal models for USH1B. They are deaf and show vestibular dysfunction. ^{11 –13} Although several shaker1 mouse models have been shown to have slightly diminished a- and b-wave amplitudes on ERG analysis, ¹⁴ retinal degeneration in shaker1 has not been observed. ^{11 –13} Therefore, it has been thought that the mouse models do not serve as a reasonable animal model system for studying photoreceptor degeneration associated with USH1. 

Photoreceptor cells have remarkable abilities to adapt to varying degrees of illumination. One of the mechanisms they use to accomplish this is the translocation of certain components in the phototransduction pathway between cellular compartments under different light/dark adaptation conditions. Under light adapted conditions, transducin will move from the outer segments to the inner segments, cell bodies, and synaptic terminals of the rod photoreceptors. Such translocation can be detected by immunocytochemistry using specific antibodies, or by serial tangential cryosectioning of the retina with immunoblotting. The detailed mechanisms regulating these translocations are still under investigation. ^{15 –17}  

Protein translocation in rods can be observed only when the light intensity exceeds a critical threshold. ¹⁸ Recent studies suggest that this threshold can be shifted to either a lower or higher light intensity, depending on the ability of the GTPase-activating complex to inactivate GTP-bound transducin. ^19,20 The threshold may be a critical point that indicates the switch of the rod from the highly light-sensitive mode that can only work under low light intensity to the less sensitive mode under high-intensity light. The protein translocation mechanism activated under a certain threshold, therefore, may serve as a neuroprotective function of rods from high-intensity light by reducing the metabolic stress generated under conditions of bright illumination. ^20,21  

Here, we report that shaker1 mice have delayed rod transducin translocation with a shift of its light activation threshold to a much higher level. Elevated threshold means that the phototransduction in the rod outer segments will continue to work in high light-sensitive mode under bright light. This may increase the metabolic stress in rods under conditions of bright illumination, and may make these rods vulnerable to light-induced degeneration. Indeed, we show that even moderate intensity light induces significant rod photoreceptor degeneration in shaker1 mice compared to strain-matched wild type mice. Moderate light exposure also leads to an accumulation of superoxide in shaker1 rods at a level 3.5 times greater than that in wild type mice. More importantly, when shaker1 are reared under a moderate light (1500 lux)/dark cycle, they develop severe retinal degeneration in less than 6 months. Shaker1 mice, when reared under the normal dim light conditions of the vivarium (<200 lux at the cage level), do not develop retinal degeneration. Our findings indicate that shaker1 mice do indeed possess a robust retinal phenotype, which has likely been masked because of low light conditions in animal housing facilities. 

Methods

Animals.

shaker1 mice (Myo7a^sh1–11J ) were purchased from Jackson Laboratories (Bar Harbor, ME). For purposes of comparison with other genetic mouse models for Usher syndrome, these mice were back-crossed 9 generations onto the pigmented 129 Sv/J background. Strain-matched wild type pigmented 129 Sv/J mice were used as controls for all studies. The RPE65 transcript for this strain was amplified and sequenced and found to be of the L450 genotype ^22,23 in both shaker1 mice and control wild type mice. The animals were kept at the Boys Town National Research Hospital vivarium in transparent cages under a 12-hour light/dark cycle. Procedures for handling animals followed National Institutes of Health guidelines and in accordance to an approved Institutional Animal Care and Use Committee protocol. Every effort was made to minimize discomfort and distress. 

Antibodies.

Antibodies against the α subunit of rod transducin (CytoSignal, Irvine, CA) were used. 

Light/Dark Adaptation.

For dark adaptation, the animals were kept in cages without any restraint or pain in a lightproof darkroom without any detectable light. For light adaptation, the animals were first kept in darkroom for 6 hours of dark adaptation, and then the animals were kept in transparent cages under various light intensities from 10 minutes to 4 hours. Light intensity was measured inside the cage. The light sources (diffuse white fluorescent light) were placed four to six inches above the cages and beside the cages from all four sides. Four-hour dark adaptation and 1-hour light adaptation is sufficient for proteins to be translocated in wild type mouse rod photoreceptors. ¹⁸ For continuous light exposure, the animals were under light or dark adaptation in their cages without any restraint or pain. The animals were first kept in darkroom for dark adaptation for 6 hours, and then the animals were kept continuously in transparent cages under 2500 lux diffuse white fluorescent light for 6 days. For rearing under long-term light/dark cycle—the animals were under 12 hours 1500 lux light/12 hours dark cycle in their cages without any restraint or pain. 

Immunocytochemistry.

This method has been described in detail in previous publications. ^{24 –26} Briefly, eyes were quickly removed from animals killed under deep anesthesia (2,2,2-tribromoethanol at a dose of 300 μg/g body weight [Avertin; Sigma]). After removal of the anterior segments, the posterior eyecups were fixed in 4% paraformaldehyde in 100 mM sodium phosphate buffer (PB; pH 7.3) at 4°C. The concentration of paraformaldehyde and time of fixation varied according to the immunoreactivities of various antigens. The tissue was transferred sequentially into 5% and 30% sucrose in PB, each at 4°C overnight. Retinal sections (2–8 μm thick), were cut with a Micron cryostat and mounted on gelatin-coated slides. Retinal sections were then incubated with 5% normal goat serum (Vector Laboratories; Burlingame, CA) in PBS for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C [anti-transducin α1 antibody (Cytosignal) 1:1000], and followed by three washes in PBS of 15 minutes each. The sections were then incubated with Alexa 594-conjugated anti-mouse immunoglobulin antibody (Invitrogen; Eugene, OR) 1:250 for 2 hours at room temperature. Staining reactions were terminated by washing with PBS and the slides were coverslipped with 50% glycerol in PBS for viewing under a Zeiss confocal microscope. All incubation and wash buffers contained Triton X-100 (0.3%). 

Counting Total Photoreceptor Number.

The number of nuclei in the outer nuclear layer (ONL) of retinal sections at different time points was calculated. At each time point, number was counted from retinal sections in central areas 2 mm eccentric from the optic nerve head site, which can be recognized under light microscopy. The central one-third of the entire length of retinal cross section traversing the entire retina width passing through the optic nerve head from superior ora serrata to inferior ora serrata was defined as the central region. Both vertical and horizontal sections across this site were examined. Serial retinal sections (4–5 μm each section for 12 sections) were taken. Mean photoreceptor numbers on these sections were counted. Only well oriented sections with straight rod outer segments that did not have oblique orientation were used. All data obtained from animal models were compared with that from the wild type retina of the same eccentricity and ages. 

Hydroethidine-Based Detection of Intracellular Superoxide Production.

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence. ^27,28 In the present study, we show that superoxide generated in light adaptation oxidizes HE to a fluorescent product in photoreceptors. The cell permeant probe HE is oxidized by superoxide to a fluorescent product, ethidium (Et). Et is retained intracellularly, allowing semiquantitative estimations of cellular superoxide production. HE (Invitrogen) was prepared as a 10-mg/mL stock in dimethylsulfoxide and stored at −20°C. Working stocks (1 mg/mL) were made in distilled water and freshly prepared. For estimation of cellular superoxide production in retina, the retina was isolated from the eyecup into culture medium. HE was added to the culture medium with the isolated retina to a concentration of 5 mg/mL and incubated for 30 minutes. The retina was then washed three times in PBS and fixed in 4% paraformaldehyde and processed for cryosectioning. Cellular Et fluorescence was measured using a fluorescence microscope (Zeiss). Before each experiment, a background image was taken that was later subtracted from the images. Fluorescence intensity data were normalized through standardization of loading procedures, background subtraction, and randomization of the experiments. 

Statistical Analysis.

All statistically analyzed data were subjected to the Student's t-test with Bonferroni correction. 

Results

Delay of Rod Transducin Translocation in Shaker1 Mouse Model.

Delay in light-induced transducin translocation in rod photoreceptors was observed in the retina of shaker1 mice (Fig. 1). In shaker1 mice, after dark adaptation for 6 hours, transducin is concentrated in the outer segments of rod photoreceptors (Fig. 1C) as is that in strain/age matched wild type mice (Fig. 1A). In the wild type mouse retina, after 1 hour 1500 lux light adaptation, almost all transducin is translocated to the inner segments, cell bodies, and synaptic terminals of rod photoreceptors (Fig. 1B). In the shaker1 mouse retina, after 1 hour 1500 lux light adaptation, the strongest immunostaining of α-transducin (yellow color) was still in the outer segments of the rod photoreceptors, and the synaptic terminals were only weakly labeled (Fig. 1D), indicating the light-induced transducin translocation in shaker1 rods was defective. Transducin in shaker1 rods did move to the inner segments and synaptic terminals, suggesting that the translocation was delayed, but not completely blocked, because after dark adaptation, similar to that in the wild typed mice (Fig. 1A), transducin in shaker1 mice was localized almost completely in the rod outer segments (Fig. 1C). After 1500 lux light adaptation for 1 hour, in shaker1 mice, the regions of rod inner segments, the outer nuclear layer and the outer plexiform layer showed weak staining of transducin (Fig. 1D) indicating some of the transducin had been translocated to the rod inner segments, cell bodies, and synaptic terminals, even though the majority of transducin staining remained in the rod outer segments (yellow color). In shaker1 mice, it took more than 5 hours of light exposure to translocate most transducin from the rod outer segments to the inner parts of the rod photoreceptors (data not shown). These results suggest that, after prolonged light exposure, most of the transducin in shaker1 rods can, in fact, be translocated to the inner segments and synaptic terminals, but the speed of translocation is much slower than that in the wild type mice, indicating the rod transducin translocation in shaker1 is delayed, but not blocked. This translocation delay could be detected in different ages of shaker1 mice tested (from 3-week-old to 1-year-old), showing it to be an inherent property of photoreceptors in the shaker1 mouse. 

Figure 1.

 

Shaker1 mice show delayed rod transducin translocation. Immunostaining of transducin α subunit on wild type (A and B) and shaker1 (C and D) retinas after dark adaptation for 6 hours (A and C) and light adaptation (1500 lux) for 1 hour (B and D). Arrows indicate transducin labeling at the rod synaptic terminals. After 1 hour of light exposure, rod synaptic terminals in a wild type mouse (arrows in B) show a very high intensity of transducin labeling, suggesting that a significant amount of transducin has been translocated to the rod synaptic terminals (the yellow labeling in Fig. 1 represents the regions with the highest intensity of transducin immunostaining). Under the same condition, after 1 hour of light exposure, the rod synaptic terminals in a shaker1 mouse (arrows in D) show only very weak labeling of transducin, while the strongest labeling of transducin (yellow color) remains at the rod outer segments, indicating the translocation of rod transducin in shaker1 is delayed. RPE, retinal pigment epithelium; PRL, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; IS, inner segments. Arrowheads indicate labeled blood vessels. Scale bar: 25 μm.

The Delay of Rod Protein Translocation in shaker1 may be Caused by a Shift of its Activation Threshold.

Protein translocation in rods can be observed only when the light intensity exceeds a critical threshold level. ¹⁸ We have found that the activation threshold of rod transducin translocation in shaker1 retinas has been shifted to a higher level. As shown in Fig. 2, after 200 lux light adaptation for 10 minutes, a significant amount of transducin in the wild type retina had already been translocated to the inner segments (large arrows in 2A) and synaptic terminals (small arrow in 2A). Under the same conditions in the shaker1 mouse retina (Fig. 2B), however, most of the transducin was still in the rod outer segments. There was little staining in the rod inner segments, and there was no staining of transducin in the synaptic terminals (OPL). When the light intensity was increased to 500 lux for 10 minutes, a significant amount of transducin in the wild type retina had moved to the inner segments and synaptic terminals (Fig. 2C). In shaker1 mice, there was still very little transducin translocated to the inner segments under these same conditions (Fig. 2D). When the shaker1 rods were light adapted for 10 minutes at 700 lux, transducin was translocated to the inner segments (large arrows in Fig. 2F) and synaptic terminals (small arrows in Fig. 2F), indicating that the activation threshold in shaker1 mice was elevated to 700 lux compared to 200 lux for wild type retinas. 

Figure 2.

 

Activation threshold for rod transducin translocation in shaker1 has been shift to 700 lux. Immunostaining of rod transducin on wild type (A, C, and E) and shaker1 (B, D, and F) retinas after light adaptation for 10 minutes of 200 (A and B), 500 (C and D), and 700 lux (E and F), respectively. Large arrows indicate labeling of transducin in rod inner segments. Small arrows indicate labeling of transducin in the rod synaptic terminals. Arrowheads indicate labeled blood vessels. Other labels are the same as in Fig. 1.

Continuous Moderate Light Exposure Induces Rod Photoreceptor Degeneration in shaker1 Mice.

It is well established that bright light can induce photoreceptor degeneration. ²⁹ However, moderate light exposure (<2500 lux) usually will not induce significant rod degeneration in pigmented wild type mouse retinas. ^29,30 We found that photoreceptors in pigmented shaker1 mice were more sensitive than strain-matched pigmented wild type mice to moderate light-induced damage. As shown in Fig. 3, continuous moderate light exposure can also induce minor rod degeneration in pigmented wild type mice, but it is much less severe than that in shaker1 mice. After a continuous (6-day) moderate light exposure (2500 lux), only less than 10% of photoreceptors were degenerated in pigmented wild type mice (average of eight wild type mice). Under the same conditions, the number of photoreceptors in shaker1 mice was reduced by more than 30% (average of eight shaker1 mice), indicating that moderate light exposure can induce much more rod degeneration in shaker1 compared to wild type mice (Fig. 3). 

Figure 3.

 

Continuous light exposure induces more rod degeneration in shaker1 mice. A and B, Light micrographs of central retinal sections of a 3-month-old control wild type mouse (A) and a 3-month-old shaker1 mouse (B) after 6 days of continuous 2500-lux light exposure. Labels are the same as in Fig. 1. C, Average densities (N = 8) of photoreceptors in the ONL of central retinal cross-sections of 3-month-old wild type (WT) and 3-month-old shaker1 mice after 6 days of continuous 200- and 2500-lux light exposure (LA). P value for WT2500lux/Shaker2500lux < 0.001.

Shaker1 Mice Reared Under Moderate Light/Dark Cycle Develop Rapid and Severe Retinal Degeneration.

When shaker1 mice were reared in vivarium rooms under low intensity light as daylight (usually <200 lux inside the cage) for light/dark cycle, they did not develop retinal degeneration even at the age of 15 months (as shown in Fig. 4 [yellow line for WT]). When 1-month-old shaker1 mice were exposed to 1500 lux light for 12 hours each day (1500 lux at the cage level, 12-hour light/dark cycle), their retinas developed significant photoreceptor degeneration in 3 months (the average rod number had reduced approximately 20%–25% in four shaker1 mice). At the age of 6 months, more than 40% of their rods (average of four shaker1 mice) had degenerated (Fig. 4C red line for shaker1L). Under the same conditions, the strain-/age-matched pigmented wild type mice did not develop appreciable degeneration (Fig. 4A and in blue line in 4C for WTL). These results show that shaker1 mice do develop retinal degeneration when they were reared under light conditions that approximate normal room-light intensity. Strain-matched wild type mice reared under the same lighting condition did not develop retinal degeneration, further suggesting that shaker1 mice are susceptible to light-induced photoreceptor degeneration even under moderate room-light. These data show that shaker1 mouse models do indeed manifest a retinal phenotype that can be revealed by manipulating light levels in the environment, and are much more sensitive to light induced photoreceptor degeneration than strain-/age-matched wild type mice. 

Figure 4.

 

A and B, Light micrographs of central retinal sections of a 9-month-old control wild type mouse (A) and a 9-month-old shaker1 mouse (B) after being reared for 6 months under a 1500-lux light/dark cycle. Labels are the same as in Fig. 1. C, Kinetics of rod loss as a function of age in wild type and shaker1 mouse retinas reared under regular vivarium room light (<200 lux at the cage level, yellow line for WT, and black line for shaker1) compared with that reared under a 1500-lux light/dark cycle (blue line for WTL, and red line for shaker1L). Data points represent quantitative measures of rod numbers (average of 4 mice) in the central parts of the retinas. *Statistically significant differences between wild typeL and shaker1 L mice (P < 0.001).

Accumulation of Superoxide in Light Adapted shaker1 Retinas.

The elevated protein translocation threshold suggests that phototransduction in the rod outer segments will continuously function in the high light-sensitive mode under bright light. This would be expected to increase the metabolic stress in rods under conditions of bright illumination. We examined oxygen-free radical accumulation in retinas from wild type and shaker mice after 6 days of continuous exposure to 2500-lux light conditions. Retinas were stained with DHE, which is converted to a fluorescent product by superoxides. Figure 5 shows that, after light adaptation for 6 days, DHE fluorescence indicated that the superoxide accumulation in the rods of shaker1 mouse retina (Fig. 5B) was significantly increased when compared with the retinas from strain-matched wild type mice under the same conditions (Fig. 5A). The average number of DHE positive photoreceptors in four shaker1 mice was approximately 3.5 times greater than that in the wild type retinas (Fig. 5C). 

Figure 5.

 

Accumulation of superoxide in light adapted shaker1 retinas. Results of DHE experiments on wild type (A) and shaker1 (B) mouse retinas after continuous 2500 lux light adaptation for 6 days. Labels are the same as in Fig. 1. C, Comparing the average number of DHE-positive photoreceptors in four shaker1 and four wild type retinas. *Statistically significant differences between wild type (WT) and shaker1 (Shak) mice (P < 0.001).

Discussion

Usher syndrome Type IB is characterized by profound congenital deafness and early onset of RP. ³¹ It is caused by mutations in the gene encoding myosin VIIa. ^8,32 Shaker1 mice have mutations in myosin VIIa. These mice are congenitally deaf, have vestibular areflexia, and are widely accepted as a mouse model for USHIB. ^11,14,33 Interestingly, even though shaker1 mice show consistent inner ear defects, they do not develop retinal degeneration, ^{11 –13,32,34,35} which has led to a widely accepted view that the Shaker1 mouse is an inappropriate species for modeling retinal pathology associated with Usher syndrome. Our results confirm that when pigmented shaker1 mice were reared under normal vivarium light intensity (<200 lux at inside cage level) for a 12-hour light/dark cycle, they do not develop appreciable photoreceptor degeneration even at the age of 15 months. However, under even moderate light conditions, shaker1 mice were more sensitive to light-induced photoreceptor damage when compared with strain-/age-matched wild type mice. Moderate light exposure does not cause significant photoreceptor degeneration in pigmented wild type mice in normal conditions. ^29,30 After 6 days of continuous 2500-lux light exposure, significant rod degeneration was observed in pigmented shaker1 mice, but not in strain-/age-matched wild type mice. More importantly, when shaker1 mice are reared under a moderate light (1500 lux)/dark cycle, they develop severe retinal degeneration in less than 6 months. This light/dark exposure approximates what is experienced in everyday life for Usher patients. Our findings indicate that shaker1 mice may possess a robust retinal light damage phenotype. It should be noted that the mice used in these studies were on the 129 Sv/J background, which harbor the L450 quantitative trait locus for RPE65 (based on our own sequencing results), and are therefore inherently more sensitive to light-induced photoreceptor cell damage than the mice with L450M in their RPE65, which have much higher resistance to light induced damage. ^22,23,36 These data may not be reproduced under the light conditions used in strains that are L450M for RPE65, which would be less sensitive. 

Why shaker1 mice are more sensitive to light-induced damage is unclear. Previous reports indicate that even though shaker1 mice do not show retinal degeneration, there are abnormalities in shaker1 retina. ERG studies indicated that, after dark adaptation for 30 minutes, both a- and b-wave amplitudes were reduced when the maximum light intensity stimulation was used. Such anomalies can be recorded from shaker1 mice at the age of postnatal day 20 to 1 year when there was no sign of reduced rod numbers, indicating it is an intrinsic defect of photoreceptors and not related to the degeneration of rods. ¹⁴ The cause of the reduced ERG waves in shaker1 mice has remained unclear. Our results show that shaker1 mice have a delay in rod transducin translocation. The translocation defect is also age-independent, and is therefore intrinsic to photoreceptor function in these mice. Whether there is any relation between reduced ERG amplitude and delayed rod protein translocation remains an important unanswered question. 

The function of protein translocation in photoreceptors is still under investigation. It is likely that α-transducin translocation is an important way for photoreceptors to adapt to various light conditions. ^15,17 Diseases with defects in light-dependent photoreceptor protein translocation associated with various functional defects, including protein transport, often show retinal degeneration. ^{37 –41} Interestingly, among these cases, there were reports that defects in protein translocation could increase the susceptibility of light-induced photoreceptor degeneration. ^37,38 It is not known how a delay in rod transducin translocation can increase the susceptibility of light-induced photoreceptor degeneration. We show that light also induces the accumulation of oxygen-free radicals in shaker1 photoreceptors, suggesting that delayed translocation may be associated with elevated susceptibility to light-induced oxidative damage. Transducin is responsible for the activation of phosphodiesterase in the phototransduction pathway. In normal conditions, when light stimulation reaches a certain threshold intensity, ¹⁸ transducin is moved out of the outer segments to reduce the activation of the phototransduction pathway. Protein translocation in rods, therefore, can be observed only when the light intensity exceeds a critical threshold level. ¹⁸ The threshold may be a critical point that activates the switch from the highly light-sensitive mode that can only work under low light intensity to the less-sensitive mode operated under high-intensity light. The protein translocation activated under certain thresholds, therefore, may be a neuroprotective function of rods that prevents damage from high-intensity light. Translocation may function to reduce the metabolic stress in rods under conditions of bright illumination and prevent constant activation of saturated rods under strong light intensity. ^20,21  

Our results show that the threshold for light-activated transducin translocation in shaker1 retina has been shifted to a higher level. This may increase the metabolic stress in rods under conditions of bright illumination, and may make these rods vulnerable to light-induced degeneration. Prolonged activation of the phototransduction cascade has been associated with photoreceptor degeneration. ^{37,39 –41} The finding of threshold shift in shaker1 rods may provide a clue for deciphering the possible molecular and cellular mechanism of how defective rod protein translocation can increase susceptibility of light-induced photoreceptor degeneration. Because the activation threshold of shaker1 has shifted to 700 lux, transducin in shaker1 rods will be translocated only when the light intensity is higher than 700 lux. When shaker1 mice were exposure under bright light higher than 200 lux, transducin in their rods cannot be translocated out of the outer segments as that in normal wild type mice because of the shift of the activation threshold. Constant light exposure even at a moderate room-light level would therefore increase the accumulation of oxygen-free radicals in shaker photoreceptors with elevated metabolic stress. 

Our most recent studies show that subretinal injection of lentiviral vectors expressing myosin VIIa can restore the transducin translocation phenotype in shaker1 mice and protect the photoreceptors from light-induced damage, showing that these phenotypes are caused by the lack of myosin VIIa function (Zallocchi M, et al. IOVS 2011;52:ARVO E-Abstract D1136). It is not known how mutations in MYO7A lead to defective transducin translocation. In vertebrate retina, myosin VIIa is reported to be found in the RPE and photoreceptors. In the RPE, it is present at the apical processes. ^{34,42 –44} In the photoreceptors, it is present at the connecting cilium region and the synapses. ^35,42,43,45 Myosin VIIa has been reported to be associated with melanosomes in RPE, and shaker1 mice have been reported to show mislocalization and defective motility of melanosomes in RPE. ^9,32 It has also been reported that replacement of MYO7A to the RPE of MYO7A-null shaker mice using lentiviral gene replacement therapy could restore the normal apical location of melanosomes in RPE cells. ¹⁰ RPE cells play critical roles in determining the life and function of photoreceptors. Defects in RPE cells can lead to photoreceptor degeneration ⁴⁶ and defective rod protein translocation. ^47,48 Therefore, an alternative explanation of the data may be related to earlier studies showing redistribution of melanosomes in the RPE in shaker 1 mice. ^9,32 The change in threshold may be caused by events in the myo7a^-/- RPE. As myosin VIIa transports melanosomes to the apical processes, the absence of the myosin motor may cause downregulation or misplacement of melanosomes. Changes in melanosomes may alter the effective light intensity in the retina and affect the threshold of light-dependent protein translocation. Furthermore, melanosomes have been shown to act as effective antioxidants, ⁴⁹ and some of antioxidant properties may be lost in shaker1 mice, explaining the accumulation of superoxide in light-adapted shaker1 retinas. 

It is conceivable that the behavioral issues with the shaker1 mice could influence the level of light exposure in our studies, thereby trivializing the results we present regarding light-induced retinal degeneration. Body weights of shaker1 mice were not statistically different from age- and sex-matched wild type mice (data not shown), suggesting that the hyperactivity observed occurs primarily when the animals are disturbed and not during the bulk periods of light exposure. In addition, more recent studies in our laboratory show that the subretinal injection of lentiviral vectors expressing myosin VIIa can rescue the sensitivity to light-induced retinal degeneration in shaker1 mice (Zallocchi M, et al. IOVS 2011;52:ARVO E-Abstract D1136). If the phenotype was simply caused by behavioral differences between shaker1 mice and strain-matched wild type mice, rescue would not be possible. 

Finally, our results suggest that light exposure may play a role in the development of RP in USH1B patients. Investigation of this question will provide information to determine whether light exposure is a significant contributor to the progression of RP associated with Usher syndrome, and will also help to determine whether reducing light exposures may delay RP development in Usher syndrome. This could lead to important clinical recommendations that might result in significant delay the onset of retinal degeneration in humans with the disease. However, it is important to note that mice are nocturnal while humans are diurnal. Nocturnal mice are much more sensitive to light-induced damage than humans. The extrapolation of our findings in mice to the human disease must be considered with caution. 

Footnotes

 Supported by a Center of Biomedical Research Excellence award through the National Institutes of Health P20 RR018788 (YWP), National Institutes of Health Grants R01 DC04844 and R01DK55000 (DC), and the Nebraska Tobacco Settlement Biomedical Research Fund.

Footnotes

 Disclosure: Y-W. Peng, None; M. Zallocchi, None; W-M. Wang, None; D. Delimont, None; D. Cosgrove, None.