Skip Navigation


Human Molecular Genetics Advance Access originally published online on July 14, 2004
Human Molecular Genetics 2004 13(18):2075-2087; doi:10.1093/hmg/ddh211
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/18/2075    most recent
ddh211v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (21)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ding, X.-Q.
Right arrow Articles by Naash, M. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ding, X.-Q.
Right arrow Articles by Naash, M. I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, Vol. 13, No. 18 © Oxford University Press 2004; all rights reserved

The R172W mutation in peripherin/rds causes a cone–rod dystrophy in transgenic mice

Xi-Qin Ding1, May Nour1, Linda M. Ritter2, Andrew F.X. Goldberg2, Steven J. Fliesler3,4 and Muna I. Naash1,*

1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA, 2Eye Research Institute, Oakland University, Oakland, MI 48309, USA, 3Department of Ophthalmology and 4Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St Louis, MO 63104, USA

Received May 10, 2004; Revised June 24, 2004; Accepted July 1, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peripherin/rds (P/rds) is a membrane glycoprotein essential for the photoreceptor outer segment disc morphogenesis and maintenance. More than half of the disease-causing mutations in P/rds have been linked to different forms of macular dystrophy; the most common one is substitution of tryptophan for arginine at position 172 (R172W). Here we confirm the patient phenotype associated with the expression of R172W mutation in transgenic mice. Functional, structural and biochemical analyses showed that, while R172W P/rds is appropriately localized, a direct correlation exists between transgene expression levels and the onset/severity of the phenotype. In the wild-type background, both cone and rod photoreceptors' structure and function were significantly diminished, which indicates a dominant-negative, cone–rod defect. Whereas rds+/– mice maintained the normal cone function at early ages, cone responses in R172W/rds+/– mice were diminished to 41% of the wild-type level signifying a preferential damaging effect of the mutation on cones. Conversely, R172W/rds+/– mice showed a significant rescue of rod function and improvement of rod outer segment structure. Although rds–/– mice have no detectable rod or cone responses, R172W/rds–/– animals retained 30% of wild-type structure and rod function, but no significant rescue of cone function was detected at 1 month of age. No biochemical abnormalities were observed in complex formation and association with Rom-1; however, R172W protein was more sensitive to tryptic digestion, indicative of a change in protein conformation, possibly contributing to the cone-dominated phenotype. As the first animal model for P/rds-associated cone–rod dystrophy, R172W mice provide a valuable tool for studying the pathophysiology of P/rds-associated human retinal dystrophies and the development of therapeutic strategies to intervene in these diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The tetraspanning trans-membrane protein peripherin/rds (P/rds) is located along the disc rim region of retinal photoreceptor outer segments (OSs) (1,2). This protein is critical for disc morphogenesis, orientation, stability and OS renewal (1,2). In retinal degeneration slow (rds) mice, a naturally occurring null mutant of P/rds, OS membrane biogenesis occurs; however, disc membrane morphogenesis is aberrant and the photoreceptors subsequently die (3–6). P/rds forms complexes with itself and with Rom-1, a non-glycosylated homologue, to form a mixture of core homo- and heterotetramers that can be linked together through intermolecular disulfide bonds to form octamers and higher-order oligomers (2,7). The C-terminus of P/rds has been shown to promote membrane fusion in vitro, signifying a possible role for this protein in OS renewal (8,9). Furthermore, P/rds has been shown recently to associate with the rod-specific, glutamic acid- and proline-rich region of the cGMP-gated channel (GARP) (10). This association is thought to play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel and the Na+/Ca2+/K+ exchanger to the plasma membrane.

Interest in P/rds has increased since the discovery of its association with different forms of human retinal diseases. Over 80 different pathogenic mutations have been identified that are associated with retinitis pigmentosa (RP), cone–rod dystrophy, pattern macular dystrophy (MD), adult vitelliform MD and central areolar choroidal dystrophy (11, http://www.sph.uth.tmc.edu/RetNet and http://www.retina-international.org/sci-news/rdsmut.htm). Wells et al. (12) identified a P/rds mutation in codon 172, which replaces an arginine with tryptophan in patients with MD. Later, patients with this mutation were reported in additional families of English, Japanese, Swiss and Spanish origins, with similar patterns of retinopathy (13–18). In addition, other mutations at position 172, including R172G and R172Q, have been found to associate with MD (14,17). Although clinical findings suggest a cone-dominant defect in patients expressing the R172W mutation, one study has shown patients to present symptoms with a more diffuse and progressive retinal degeneration (19). Molday et al. (20) expressed P/rds with this mutation in an in vitro system. The R172W protein appeared normal with respect to dimerization, glycosylation and subunit assembly with itself and with Rom-1. They predicted that the R172W mutation causes a subtle change in the structure of P/rds that affects its function in cone, but not in rod, photoreceptors.

Transgenic mouse models of human retinal diseases have been widely used for studying the effects of genetic mutations on cellular structure and function (21–25). To investigate the pathogenic defects and the mechanism of disease caused by the R172W mutation, transgenic mice expressing the mutant protein in both rods and cones were generated and their retinas were characterized structurally, functionally and biochemically. In all rds genetic backgrounds, R172W mice initially presented with profound cone defect, followed by rod abnormalities, characteristic of cone–rod dystrophies. Although no biochemical changes with regard to either complex formation or interactions with Rom-1 were detected, the R172W protein was found to be more susceptible to limited tryptic digestion, indicative of a change in protein conformation, which may be a contributing factor in the cone-dominant pathogenic effects.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
R172W protein localizes properly to rod and cone OSs
To evaluate the effects of the R172W mutation on retinal structure and function, transgenic mice expressing the mutant protein under the control of the human interphotoreceptor retinoid binding protein (hIRBP) promoter were generated (Fig. 1A). This heterologous promoter has been previously shown to successfully direct transgene expression to both rods and cones (26–35). Thirteen permanent lines with a single site of integration were generated and characterized for the pathogenic effect of the mutation in relation to the transgene expression level. Figure 1B shows the R172W protein level from two of these lines that were considered as low and high expresser lines in the absence of the endogenous P/rds. R172W protein migrated at the 36 and 78 kDa markers, corresponding to the wild-type P/rds monomers and dimers, respectively (Fig. 1B, left). Densitometric analysis of these bands revealed the presence of 40 and 75% of the wild-type P/rds in R172W+/+ retinas taken from the low and high expresser lines on the rds–/– background, respectively (Fig. 1B, right).



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Expression and localization of R172W protein. (A) A schematic illustration of the R172W transgene construction. (B) Western blot analysis of P/rds from 1-month-old wild-type and R172W retinas of the indicated genotypes. The left panel is a typical western blot of P/rds from wild-type and two R172W lines. The right panel shows densitometric measurements of protein levels. Data represent mean±SEM for three independent experiments, each performed on retinas combined from four to six animals. (C) Immunofluorescence localization of R172W P/rds in the OSs of photoreceptors. R172W protein in the OSs was detected by FITC 3B6 (a). Blue cone cells were labeled by the anti-blue cone opsin antibody (b). Merged images confirmed the cone localization of the mutant protein (c). Using laser deconvolution microscope at high magnification revealed the disc rim localization of the mutant proteins in both rods (d) and cones (e and f), as indicated by arrow and arrowhead, respectively.

 
The monoclonal antibody 3B6 (3B6 mAb) (36,37) was used to determine the cellular localization of R172W protein. To allow for 3B6 recognition of the R172W protein, but not the endogenous mouse P/rds, the 3B6 epitope was constructed in the C-terminal region of the transgene by substituting the proline normally present at position 341 with glutamine (P341Q). In a separate study, we have evaluated the side effects of P341Q modification on P/rds function in rods and cones using the same transgene design employed for the R172W mutation (38). Transgenic lines carrying the P341Q modification alone are named ‘NMP’ (normal mouse P/rds). Characterization of ‘NMP’ retinas on all rds genetic backgrounds demonstrated that the P341Q modification causes no alterations in the biochemical or functional properties of P/rds, nor any adverse effects on retinal structure (38). Furthermore, P/rds bearing the P341Q modification was able to rescue the rds phenotype in a dose-dependent manner (38). It is important to note that the 3B6 mAb affords selective recognition of P/rds bearing the C-terminal P341Q modification in NMP retinas by immunohistochemistry, but not the endogenous P/rds protein in wild-type retinas (38).

Double immunofluorescence staining with 3B6 and anti-blue cone opsin antibodies in retinal sections taken from 1-month-old R172W mice is shown in Fig. 1C. Labeling with 3B6 mAb shows exclusive localization of R172W protein to photoreceptor OSs (Fig. 1Ca). Co-localization of 3B6 and anti-blue opsin labeling confirms the presence of R172W protein in cone photoreceptor OSs (Fig. 1Cc). Similar results were observed with the anti-red/green opsin antibody (data not shown). At higher magnification, using laser deconvolution microscopy, localization of R172W protein was confirmed to be in the disc rim region of photoreceptors (Fig. 1Cd). An overlay image showing co-localization of immunostaining with 3B6 and anti-blue opsin antibodies further demonstrates disc rim localization of R172W protein in cone photoreceptors (Fig. 1Cf). This immunoreactivity was cell type-specific, as no additional labeling was found in any other retinal cell type or cell layer.

R172W mutation causes a dominant-negative cone–rod dystrophy
We had previously described a mild, late-onset cone defect in the low expresser line in which cone functional deficits were concomitant with cone photoreceptor degeneration (39). In the present study, we have focused on the phenotype in the high expresser line in which homozygotes express P/rds at a level equivalent to ~75% of the wild-type.

To determine whether the R172W mutation causes a dominant-negative phenotype, transgenic retinas from the high expresser line were characterized in the presence of two wild-type alleles. Retinal cross sections from R172W+/+ on the wild-type background at 1 month of age showed mild OS disorganization and reduction in OS length, but without loss of photoreceptor nuclei (Fig. 2A, upper panel). Ultrastructural (EM) analysis further demonstrated the R172W-mediated disruption of OS structure (Fig. 2A, arrows in the lower panel). Functional studies of these animals showed a significant reduction in both scotopic (rod) and photopic (cone) electroretinograms (ERG) as early as 1 month of age, and photoreceptor function continued to deteriorate in an age-dependent manner (Fig. 2B and C). Scotopic a-wave amplitudes were reduced to 60 and 46% of wild-type levels at 1 and 2 months of age, respectively (Fig. 2B). Interestingly, an even more profound reduction was detected in the corresponding photopic b-wave responses, where transgenic mice retained only 25 and 23% of wild-type level at 1 and 2 months of age, respectively (Fig. 2C). These results demonstrate a more severe effect of the R172W mutation on cone than on rod photoreceptors.



View larger version (98K):
[in this window]
[in a new window]
  		Figure 2. Dominant-negative effect of R172W protein in the wild-type background. (A) Light (upper panels) and electron (lower panel) microscopic images of retinal sections taken from wild-type and R172W mice at 1 month of age. (B) Rod scotopic and (C) cone photopic ERG analysis of wild-type and R172W mice at 1 and 2 months of age. Data are represented as mean ± SEM of three independent measurements from six to 10 mice. Significant differences (P<0.05, Student's t-test) between transgenic and wild-type mice are marked with asterisks.

 
R172W P/rds rescues rods but not cones of the rds+/– retina
R172W mice were crossed onto the rds+/– genetic background because mice heterozygous for the R172W transgene and hemizygous for the endogenous allele more closely mimic expression levels of the respective proteins in patients carrying this mutation. Several studies have previously described a haploinsufficiency phenotype in rds+/– mice (25,38). In the absence of one endogenous allele, histological evaluation of retinal sections shows abnormal, swirl-like morphology of photoreceptor OSs, demonstrated more clearly at the electron microscopic level (Fig. 3Ab and g). However, the addition of one allele of the R172W transgene (R172W+/–/rds+/–) affords measurable improvement of photoreceptor OS structure, which can be observed at both the light and the electron microscopic levels (Fig. 3Ac and h), even as late as 3 months of age. This structural rescue was correlated with functional rescue, as evidenced by enhanced scotopic ERG responses detected in R172W animals (Fig. 3B). The rds+/– retinas retained 38% of wild-type scotopic a-wave response, whereas R172W+/–/rds+/– mice retained 70% of wild-type level (Fig. 3B). However, in sharp contrast to this improvement in rod function, a significant reduction in photopic b-wave ERG amplitudes was observed in R172W+/–/rds+/– mice, relative to rds+/– controls, suggesting an R172W-mediated, dominant-negative effect specifically on cone photoreceptors (Fig. 3C). The rds+/– mice showed normal cone photoreceptor function, whereas the R172W+/–/rds+/– mice retained 41% of wild-type amplitudes at 1 month of age (Fig. 3C).



View larger version (105K):
[in this window]
[in a new window]
  		Figure 3. R172W protein rescues rods but not cones of rds+/– and rds–/– retinas. (A) Light (upper panels) and electron microscopic images (lower panels) of retinal sections from wild-type, rds+/– and R172W mice in the rds+/– and rds–/– backgrounds at 1 month of age. (B) Scotopic and (C) photopic ERG analysis of R172W mice on rds+/– and rds–/– background at 1 month of age. Data are represented as mean ± SEM of three independent measurements from six to 10 mice. Significant differences (P<0.05, Student's t-test) between transgenics and non-transgenics are marked with asterisks.

 
R172W P/rds rescues rods, but not cones, in the rds–/– retina
Mice homozygous for the R172W transgene were crossed onto an rds–/– (null) background to assess the ability of the R172W protein to independently support OS morphogenesis and photoreceptor function, both of which are lacking in the rds–/– retina. Retinal cross sections taken from 1-month-old rds–/– mice show the absence of OSs (Fig. 3Ad) compared with a partial rescue with OS formation in R172W+/+/rds–/– mice (Fig. 3Ae). Ultrastructural evaluation of these retinas revealed a significant restoration in both photoreceptor outer and inner segment structure when compared with non-transgenic controls (Fig. 3Ai and j); however, R172W+/+/rds–/– retinas continue to display aberrant OS structure, relative to wild-type. This improvement in retinal structure was accompanied by a partial restoration (30% of wild-type level) of the scotopic a-wave (Fig. 3B), but not the photopic b-wave amplitudes (Fig. 3C).

R172W protein complexes properly with Rom-1
P/rds and Rom-1 interact with each other to form an array of heteromolecular complexes needed for disc rim assembly and OS formation (40,41). Using reciprocal co-immunoprecipitation, we evaluated the biochemical effect of the R172W mutation on P/rds–Rom-1 associations and complex formation. The results demonstrate the association between Rom-1 and R172W protein (Fig. 4). As expected, no Rom-1 from rds–/– retinas was co-precipitated by rds-CT Ab (antibodies to wild-type P/rds). These assays were also performed in the absence of any antibodies (non-immune control) and also with an unrelated antibody (selective for cone CNG-3 protein) to determine the specificity of these interactions. No immuno-precipitates were detected with any of these controls.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Association and complex formation of R172W protein with Rom-1. Retinal extracts from wild-type and R172W/rds–/– mice were immunoprecipitated with rds-CT or Rom-1-CT antibodies, and the immunoprecipitates were resolved on 10% SDS gel, followed by western blotting with the respective antibodies. Retinal sample from rds–/– mice was included as a control. Assays in the absence of antibodies or in the presence of an unrelated antibody (cone CNG-3) were included as controls. The experiments were performed with retinas combined from four to six animals for each genotype.

 
Complex assembly with R172W protein.
Non-covalent tetrameric subunit assembly is a crucial aspect of P/rds function, mediated by determinants within the large D2 regions, and is commonly measured using a velocity sedimentation assay (42). Impaired subunit assembly in in vitro studies has been correlated with point mutations causing photoreceptor degeneration and cell loss in patients (43). We have characterized the sedimentation velocity profile of the R172W protein from transgenic mice in the rds–/– background to ask whether pathogenicity of this mutation is associated with a defect in subunit assembly.

We prepared OS membranes from freshly dissected wild-type mouse retinas and from retinas of R172W mice on the rds–/– background, and sedimented detergent extracts on sucrose gradients (42). The gradients were fractionated, and the fractions were assayed for P/rds and Rom-1 protein, using western blot analysis with the appropriate monospecific antibodies. Anti-P/rds antibody used in these experiments can recognize both the wild-type and the R172W protein. Typical data are presented in Figure 5. The total number of fractions collected varied between gradients and was taken into account for the calculation of sedimentation coefficients.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 5. Subunit assembly assay by reducing velocity sedimentation analysis of P/rds and Rom-1 from detergent solubilized mouse ROS membrane preparations. Triton X-100 extracts from crude mouse ROS were reduced with 2-ME and sedimented in 5–20% (w/w) sucrose gradients; fractionated gradients (and particulate fractions, P) were assayed by western blot analysis with anti-P/rds (A and B) or anti-Rom-1 antibodies (C and D). Chemiluminescent blots and corresponding plots generated by image analysis are shown. The total number of fractions collected varied between gradients and was taken into account for the calculation of sedimentation coefficients.

 
In a previous study (44), it was found that P/rds protein from wild-type CD1 mouse retinas sediments as a single major species, under reducing conditions, with a Svedberg coefficient (S) of 4.9±0.1 (n=4), indicating a tetrameric subunit assembly. Similarly, P/rds from retinas of wild-type animals of the background strain used here (C57BL/6) sediments in a tetrameric form, with an S-value of 5.1±0.2 (n=3). We compared the sedimentation profile of R172W P/rds to that of wild-type and found it essentially indistinguishable (Fig. 5). The sedimentation coefficient calculated for the R172W mutant, 5.0±0.3 (n=3), is also nearly identical to that of wild-type. Thus, the R172W protein, like wild-type P/rds, is properly assembled as a non-covalent tetramer. These results confirm earlier work obtained using COS-1 cells expressing the same mutant protein (43).

As the small mass difference between homo- and heteromeric P/rds tetramers is not resolved by the sedimentation velocity assay, we repeated our analysis and probed for Rom-1. As seen in Figure 5, we found comparable sedimentation profiles and calculated nearly identical sedimentation coefficients: 4.8S (average, n=2) for Rom-1 from the wild-type animals and 5.0S (average, n=2) for Rom-1 from the R172W transgenics. These values indicate that, akin to our findings in wild-type animals, Rom-1, like P/rds, is solubilized from OS membranes as non-covalently associated tetramers under reducing conditions.

Disulfide-mediated oligomerization.
Under non-reducing conditions, P/rds is extracted from rod OSs in disulfide-bonded forms, indicating that both intra- and intermolecular disulfide bonds play a role in protein structure (2,43). P/rds-containing tetramers form disulfide-mediated oligomers that appear as higher-order complexes by velocity sedimentation; this correlates with increases in their mass and sedimentation coefficients. We have employed the velocity sedimentation approach to characterize potential effects of the R172W mutation on disulfide-mediated oligomerization. When Triton X-100 solubilized wild-type mouse OS membranes are sedimented under non-reducing conditions (Fig. 6A), a relatively broad distribution of anti-P/rds immunoreactive material is observed; in contrast, a considerably more narrow peak is observed for P/rds under reducing conditions (Fig. 5). The species that migrates most slowly is consistent with a P/rds-containing tetramer; its calculated sedimentation coefficient is 4.8S. The more rapidly migrating species represent higher-order, disulfide-bonded P/rds oligomers (2). Although a considerable amount of material was seen in the particulate fraction in this experiment (~20%), we found significant interexperimental variability in the quantity of sedimentable P/rds, ranging from <5% to ~20%.



View larger version (55K):
[in this window]
[in a new window]
  		Figure 6. Assay of disulfide-dependent higher-order interactions by non-reducing velocity sedimentation analysis of P/rds and Rom-1 from detergent-solubilized mouse ROS. NEM-treated Triton X-100 extracts from crude mouse ROS membrane preparations were sedimented in 5–20% (w/w) sucrose gradients; fractionated gradients (and particulate fractions, P) were assayed by western blot analysis with anti-P/rds (A and B) or anti-Rom-1 antibodies (C and D). Chemiluminescent blots and corresponding densitometry plots generated by image analysis are shown. The total number of fractions collected varied between gradients and was taken into account for the calculation of sedimentation coefficients. Immunoreactivity is more broadly distributed relative to reduced samples (Fig. 5) and reflects disulfide-dependent interactions of tetrameric core complexes with each other and possibly other additional proteins.

 
Figure 6B shows similar data for the R172W protein from transgenic retinas in the rds–/– background. Again, a relatively broad distribution of anti-P/rds immunoreactive material is observed, with the slowest species sedimenting as tetramers (4.7S). Higher-order species and particulate P/rds are present in quantities roughly comparable to those obtained from wild-type retinas. These results suggest that the R172W mutation does not perturb disulfide-dependent P/rds oligomerization.

Analogous experiments were performed to characterize the behavior of Rom-1 in the R172W transgenic retinas. As seen in Figure 6C, Rom-1 is largely excluded from both the heaviest gradient fraction and the particulate fraction. These findings support a previous observation that Rom-1 does not participate fully in the disulfide-dependent higher-order structures documented for P/rds (2). Figure 6D shows the sedimentation profile for Rom-1 from the transgenic retinas in the rds–/– background. The distribution is qualitatively similar to that observed for Rom-1 from the wild-type retinas and suggests that the R172W mutation does not strongly perturb the incorporation of Rom-1 into disulfide-dependent oligomers.

R172W mutation causes a subtle change in the protein conformation
A change in protein susceptibility to proteolysis is one indication of differences in protein folding and/or conformation (45–47). For this reason, we used limited tryptic cleavage analysis, performed on crude OS preparations from wild-type and R172W retinas on the rds–/– background, to evaluate the possibility of mutation-dependent changes in protein conformation. As shown in Figure 7, under the given experimental conditions, a significant amount of wild-type protein was spared from tryptic digestion, even after 10 min of incubation. In contrast, R172W protein was substantially digested under the same conditions (Fig. 7A). Densitometric measurements of these western blot bands (both monomer and dimer), taken from three independent experiments, are shown in Figure 7B. After 10 min of trypsin exposure, the amount of undigested protein in the R172W sample was calculated to be ~40% of the initial amount, compared with ~80% of protein remaining in wild-type samples. In a separate study, tryptic digestion of the NMP protein was performed and compared with wild-type P/rds to exclude the possibility of contributions of the P341Q modification to the observed differences in protease susceptibility. No difference was observed between these two groups (data not shown), suggesting a specific effect by R172W mutation. The enhanced susceptibility of the R172W protein to proteolytic digestion likely reflects an alteration in protein folding.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 7. Limited tryptic digestion of wild-type and R172W P/rds. Mouse ROS preparations (30 µg protein) from wild-type and transgenic mice in rds–/– background were incubated with trypsin–TPCK at a ratio of 1 : 5 of trypsin to ROS proteins for 0, 5 or 10 min. The digested products were resolved on 10% SDS gel and transferred onto PVDF membranes, followed by western blot analysis using anti-P/rds. (A) A typical immunoblot image showing the limited proteolysis of the wild-type and mutant P/rds. (B) Densitometric analysis of data taken from three independent experiments (mean ± SEM). The experiments were performed with retinal samples combined from four to six animals for each genotype. Significant differences (P<0.05, Student's t-test) between the trypsin-treated and untreated samples are marked with asterisks.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although there are over 80 different mutations in P/rds reported to associate with retinal degenerations, only a few mutations frequently found in ADRP patients have been investigated in transgenic mice (48–50). Currently, no animal models exist that mimic the cone-specific defect caused by mutations in the P/rds gene. In this study, we generated a transgenic mouse model carrying the R172W mutation in P/rds, which previously has been linked to both cone–rod degeneration and a pure cone disease in humans (12,14,15,17,18). The R172W protein appropriately localized to rod and cone OSs and the phenotype in transgenic mice was well-correlated with the reported clinical symptoms of patients carrying this mutation. Transgenic mice from the low expresser line (40% of wild-type) showed a mild, late-onset cone dystrophy in which cone functional deficits were associated with reduction in cone density at 9 months and older (39). However, the higher expresser line (75% of the wild-type) revealed an early-onset, autosomal dominant cone–rod dystrophy. The functional and structural characteristics of the transgenic retinas from the high expresser line on different rds genetic backgrounds are summarized in Table 1.


View this table:
[in this window]
[in a new window]
  		Table 1. Functional and structural characteristics of the R172W transgenic mice
 
Taking advantage of the rds–/– mutant, we effectively modulated the ratios of the endogenous to R172W protein in vivo in order to assess the dominant effect of the mutant protein on retinal structure and function. Overall, cone photoreceptors were found to be more negatively affected by the expression of the R172W protein when compared with rods. Although homozygous transgenics in the wild-type background showed a significant reduction in the dark-adapted rod ERG as early as 1 month of age, this was accompanied by a much more striking reduction in cone ERG amplitudes. Nevertheless, it is important to note that while this mutation has been previously reported to cause macular degeneration in a number of families of different ethnic backgrounds (13–15,17–19,51), a Swedish family carrying the R172W mutation in P/rds also has been identified and diagnosed with RP, a rod-dominant disease (19). Experimental evidence strongly suggests that the defect in rod function observed in transgenic animals on the wild-type background is specific to the R172W mutation, rather than an effect of P/rds overexpression, as we have previously shown unaltered retinal function and structure in response to P/rds overexpression in the mouse retina (38).

An interesting phenotypic pattern was observed in the R172W retina when mice were crossed onto an rds+/– genetic background. This is due, in part, to the combination of haploinsufficiency phenotype reported previously (25,38) and the effect of the mutation. The absence of one endogenous allele of P/rds has both structural and functional manifestations in the mouse retina. Structurally, normally aligned OSs become disorganized and rearranged in a swirl-like pattern. This abnormality in retinal structure correlates with rod-dominant electroretinographic dysfunction. Cone photoreceptors are spared until later ages, where the deficit in rod function is followed by a slow pattern of retinal degeneration (25,38). The introduction of a single R172W allele in this genetic background dramatically altered the pattern of functional and structural defects associated with P/rds haploinsufficiency. Even as late as 3 months of age, a notable rescue in rod function and OS ultrastructure was observed in R172W transgenics when compared with non-transgenic littermates, indicating that the presence of the R172W protein is less damaging to rods than its sheer absence. In contrast to the improvement in rod function afforded by the expression of the R172W protein, cone photoreceptor function was considerably reduced in these animals. This loss in cone function previously has been shown to correlate with cone photoreceptor degeneration, as demonstrated by a reduction in cone cell number (39).

In order to determine the ability of the R172W protein to support and maintain OS morphogenesis and integrity in the absence of the endogenous P/rds, transgenics were crossed onto an rds–/– genetic background. Unlike rds–/– retinas, in which the absence of P/rds leads to a failure in OS formation (i.e. a dysplasia), comparative histological evaluation of R172W+/+/rds–/– retinas revealed a notable improvement in OS structure, albeit abnormal in length and shape in comparison to wild-type animals. Functionally, the newly formed OSs in R172W+/+/rds–/– animals produced a significant rescue in scotopic (rod), but not photopic (cone), function when compared with non-transgenic rds–/– controls. In fact, the cone photoreceptor ERG signal was barely detectable in retinas exclusively carrying the R172W protein. We have demonstrated previously that a positive correlation exists between the structural and functional integrity of the retina and wild-type P/rds expression levels (38). This, however, was not the case in terms of the R172W protein expression. In the absence of the endogenous P/rds, animals homozygous for the R172W transgene generated protein expression level equivalent to ~75% of wild-type levels. It is important to highlight the fact that, though 50% of P/rds protein expression (i.e. in rds+/– retinas) is not sufficient for maintaining rod function, this amount is adequate for supporting full cone photoreceptor function at early ages. This strongly suggests a cone-specific defect caused by the R172W mutation because transgenics exclusively expressing mutant protein at abundant amounts still fail to support any significant cone photoreceptor function.

To date, the mechanism responsible for mediating the deleterious effects of the R172W mutation on photoreceptor structure and function (particularly in cones) has yet to be elucidated. This study is the first to describe the specific molecular and biochemical properties of this mutant protein that most likely contribute to its pathogenic effects. A biochemical evaluation of the R172W protein in the absence of the endogenous P/rds (R172W+/+/rds–/– retinas) revealed a similar pattern of disulfide-linked dimer and higher-order oligomer formation as found in wild-type retinal extracts. The ability of the mutant protein to interact with Rom-1 was confirmed by reciprocal co-immunoprecipitation and showed a pattern much like that found in wild-type retinas. Additionally, the R172W mutation caused no impairment in P/rds or Rom-1 subunit assembly. Impaired subunit assembly is detected as a change in the core complex sedimentation coefficient under reducing conditions. For example, the L185P mutation linked to digenic RP was found to prevent tetrameric assembly of recombinant P/rds in transfected COS cells and resulted in a >30% decrease in S-value (20). Using the same expression system, several insertional mutations were found to prevent tetrameric assembly and cause protein misfolding and aggregation (42). Thus, the wild-type sedimentation profiles and coefficients demonstrated by the R172W mutant argue strongly that its pathogenic effects are not the result of impaired subunit assembly. These findings are in agreement with what has been shown in prior studies where R172W protein was heterologously expressed in COS-1 cells (20). Interestingly, the R172W mutation is situated between regions that are essential for protein stability and normal subunit assembly. An insertion between positions G170 and F171 causes gross protein misfolding, whereas insertions further downstream, between positions S188 and S189, merely impair subunit assembly (42). Higher resolution models of the large D2 domain will be required in order to properly understand the structural and functional ramifications of mutations in this region.

It is more difficult to draw firm conclusions regarding the effect of the R172W mutation upon disulfide-dependent incorporation of tetramers into higher-order species, both because of the difficulty in obtaining robust data and in view of a recent report of P/rds participation in more widespread protein–protein interactions (10). Although the current findings indicate that the R172W mutation does not prevent P/rds-containing tetramers from assembling into disulfide-mediated higher-order structures, it is likely however that the mutation causes a mild conformational change in the complex that is detrimental to cones, but not rods. This cone-specific defect is likely due to the differences in the cone OS structure that render cones more susceptibility to these conformational changes than rods. It is also possible that the R172W mutation interrupts an important association of the complex to an as yet identified protein that is specific to cones. These kinds of effects are hard to recognize in a rod-dominant retina. We are in the process of crossing the R172W mice onto an NRL–/– background (52), which represents a cone-dominant retina.

This study is the first to evaluate the effects of a cone-dominant mutation in P/rds using transgenic mice. Although we have shown in a separate study (38) that the P341Q exerts no structural, functional or biochemical effect on wild-type P/rds, we cannot exclude the possibility that double mutant might function differently. However, this possibility is remote because we consistently observed a cone-specific defect in all rds genetic backgrounds. This cone-specific defect associated with the R172W mutation is well-correlated with the patient phenotype where other mutations at position 172, including R172G and R172Q, have been found to associate with MD (14,17). R172W transgenic mice showed functional defects similar to those reported in patients carrying the same mutation, thus demonstrating the fidelity and suitability of these animals as models for human retinal diseases. Hence, these mice have great potential for use in elucidating the defects associated with the mutation and for evaluating future therapeutic interventions for cone–rod dystrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of transgenic mice
The full-length mouse P/rds cDNA clone was isolated by screening the mouse retinal expression library ({lambda} ZAP II) and was used to generate the R172W transgene. The transgene was directed to rods and cones by a 1.3 kb fragment of the hIRBP promoter and included a 0.7 kb SV40 small t-intron and a poly A signal. The R172W mutation was introduced by PCR-mediated and oligonucleotide-directed mutagenesis (21). The accuracy of the transgene construct was confirmed by sequencing of both strands. A P341Q modification was introduced at the C-terminus of the mutant protein to mimic that of the bovine, which can be identified by the monoclonal antibody 3B6 that recognizes an epitope in bovine, rat and human, but not mouse P/rds (36). The purified transgene was microinjected into 1-day-old embryos isolated from CD1 pregnant female mice (Charles River, Wilmington, MA, USA). Transgenic mice were identified by PCR with primers specific for the hIRBP promoter and the coding region of P/rds cDNA. Thirteen potential founders were generated, of which eight passed the transgene onto their offspring and were mated with C57BL/6 mice to generate the F1 generation. The retinal degeneration (rd) mutation carried by the CD1 strain was eliminated by out-breeding and the elimination of the rd mutation was confirmed by PCR as described before (21). Breeding schemes were designed to cross the R172W transgene onto wild-type, rds+/– and rds–/– genetic backgrounds. Mice were screened for the presence of the rds mutation using PCR and Southern blot analysis, as previously described (25). Animals used in this study were maintained on a 12 h light/dark cycle (~7 foot candles). All procedures were approved by the local Institutional Animal Care and Use Committees (IACUC) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Histology and electron microscopy
After sacrifice, the superior cornea was marked with a heated needle before enucleation and a slit was made at the limbus prior to a 1 h fixation in freshly prepared 0.1 M sodium phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde, 2.0% paraformaldehyde and 0.025% CaCl2. The cornea and lens were then removed and fixation was continued overnight and then processed as described (53). Semi-thin (0.75 µm) histological sections were stained and photographed with an Olympus BH-2 photomicroscope in the auto-expose mode, using 20x or 60x DPlanApo objectives. Thin (silver) sections were collected onto copper 75/300 mesh grids, post-stained with 2% uranyl acetate (aqueous) and Reynolds lead citrate, and photographed on a JEOL 100CX electron microscope at an accelerating voltage of 60 keV.

Immunofluorescence labeling
Eyes were prepared for immunofluorescence labeling as previously described (25). Eye sections (6 µm thickness) were incubated overnight at 4°C with primary antibodies. Anti-P/rds monoclonal antibody 3B6 (a generous gift from Dr Robert Molday, University of British Columbia, Canada) was used at a dilution ratio of 1 : 5, whereas anti-human-blue opsin (a generous gift from Dr Jeremy Nathans, Johns Hopkins University, MD, USA) was used at a dilution ratio of 1 : 3000 (54). Sections were subsequently rinsed and incubated with biotinylated FITC secondary antibody (1 : 100 dilution) for 30 min at room temperature. Following secondary antibody incubation and rinses, slides were mounted, cover-slipped, and then assessed either using a Zeiss Axioscope epifluorescence microscope (Carl Zeiss, Jena, Germany) or a Leica TCS Sp2 confocal microscope (Leica Microsystems, Wetzler, Germany).

Scotopic and photopic ERG
ERG testing was carried out as previously described (55,56). Briefly, overnight dark-adapted mice were anesthetized and their eyes were dilated using 1–2 drops of 2.5% phenylephrine in each eye (Akorn, Inc., Decatur, IL, USA). Retinal electrophysiological function was evaluated using an LKC system (Gaithersburg, MD, USA) and potentials were recorded using a stainless steel wire contacting the corneal surface through a layer of 2.5% methylcellulose. Responses were differentially amplified (half bandpass, 1–4000 Hz), averaged and stored using a Nicolet Compact 4 signal averaging system. For assessment of rod photoreceptor function (scotopic ERG), a strobe flash stimulus was presented to the dark-adapted, dilated eyes in a Nicolet Ganzfeld (GS-2000) with a 137 cd (s/m2) flash intensity. To evaluate cone photoreceptor function (photopic ERG), a strobe flash stimulus was presented to the light-adapted (5 min, 29.0285 cd/m2) and dilated eyes in a Nicolet Ganzfeld (GS-2000) with a 77 cd (s/m2) flash intensity.

Western blot and immunoprecipitation
Polyclonal antibodies against residues 331–346 of the P/rds C-terminus (rds-CT Ab) and residues 336–351 of the Rom-1 C-terminus (Rom-1-CT Ab) were used in western blot and immunoprecipitation analyses. Dissected retinas were homogenized on ice, solubilized for 1 h at 4°C in solubilization buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.05% SDS, 2.5% glycerol and 1.0 mM phenylmethylsulfonyl fluoride), and processed for western blot (30 µg protein) and immunoprecipitation (200 µg protein) analyses as previously described (38,57). As a control for binding specificity, assays were performed in the absence of any antibodies and also with an unrelated antibody against the {alpha} subunit of the cone CNG channel (CNG-3). The intensities of P/rds and Rom-1 bands were quantified relative to the wild-type band. For analysis of disulfide-linked dimers, gel electrophoresis under non-reducing conditions was conducted by omitting dithiothreitol (DTT) from the sample buffer.

Preparation of ROS extracts
Retinas from 6- to 12-week-old mice were dissected and immersed in homogenization buffer (buffer A, 20 µl/retina) containing 20 mM Tris–acetate, pH 7.2, 0.25 mM MgCl2, 8 mM taurine, 8 mM D-glucose, 20% (w/v) sucrose, Pefabloc-SC® protease inhibitor (0.4 mg/ml; Roche, Inc.), leupeptin (10 µg/ml/ml) and Pepstatin A (10 µg/ml) at 4°C under normal room lighting. ROS were dislodged by vigorous vortexing (3x30 s) and were collected as the supernatant fraction after centrifugation at 2500g for 1 min. The pellet was re-extracted with additional homogenization buffer (40 µl/retina), and resultant supernatants were combined and centrifuged again at 2500g for 1 min. The final supernatant containing the crude ROS was snap-frozen on dry ice and stored at –70°C until use.

ROS membranes were pelleted by centrifugation at 50 000g for 15 min at 4°C in a Beckman TLA-55 rotor and the pellet was resuspended in phosphate-buffered saline (PBS), pH 7.5 (35–75 µl/retina), and then solubilized by the addition of an equal volume of ice-cold detergent solution with gentle vortexing. Solubilization under reducing conditions utilized buffer R, containing 2x PBS, 2% Triton X-100, 2 mM DTT, pH 7.5. Solubilization under non-reducing conditions utilized buffer N, containing 2x PBS, 2% Triton X-100, 5 mM NEM, pH 7.5. After a 20 min incubation on ice, extracts were centrifuged at 90 000g for 30 min at 4°C in a Beckman TLA-55 rotor to remove remaining particulates. Supernatants were kept on ice until use.

Velocity sedimentation analysis
Sucrose density gradient velocity sedimentation of Triton X-100 solubilized ROS, P/rds and Rom-1 detections and quantifications by image analysis of western blots were performed under reducing conditions essentially as described previously (42). Analogous analyses under non-reducing conditions were performed by replacing the DTT in both extraction and gradient buffers with 10 mM NEM. Svedberg coefficients (S20,w values) were calculated using the partial specific volume value ({nu}=0.83 ml/g) determined previously for the P/rds–Rom-1 complex from bovine ROS preparations (40) and TLS-55 rotor dimensions described in the previous study (58). In each fraction, the immunoreactivity of P/rds and Rom-1 was assessed by western blot analysis using rds-CT Ab and Rom-1 CT Ab, as previously described (42).

Limited tryptic digestion
Limited tryptic digestion experiments were performed as previously described (45,46). Mouse ROS membranes (30 µg protein) from wild-type and transgenic retinas were resuspended in protease inhibitor-free buffer A and incubated with trypsin–TPCK (Sigma-Aldrich, St Louis, MO, USA) at a ratio of 1 : 5 of trypsin to ROS membrane proteins at 30°C for various periods of time. The trypsin-treated samples were then solubilized with 2x Laemmli sample buffer and resolved on 10% SDS gels, transferred onto PVDF membranes for immunoblot analysis using anti-P/rds antibody.


   ACKNOWLEDGEMENTS
 
We wish to thank Dr Chibo Li for construction of the transgene, Barbara Nagel for excellent technical assistance in the course of the histological and ultrastructural analyses, Alexander Quiambao and Dr Ming Cheng for their exceptional technical assistance on animal screening and breeding strategies. We also thank Drs Robert S. Molday (University of British Columbia, Vancouver, Canada) and Jeremy Nathans (Johns Hopkins University School of Medicine, Baltimore, MD, USA) for generously providing mAb 3B6 and antibodies against human blue cone opsin, respectively. We are grateful to Dr Muayyad R. Al-Ubaidi, Rafal Farjo and Zack Nash for their helpful comments on the article. This study was supported by grants from the National Institutes of Health [EY10609 (M.I.N.), EY13246 (A.F.X.G.) and EY07361 (S.J.F.), and Core Grant for Vision Research EY12190 (M.I.N.)], by the Foundation Fighting Blindness (M.I.N.), and by an unrestricted departmental grant from Research to Prevent Blindness (S.J.F.). M.I.N. is a recipient of the Research to Prevent Blindness James S. Adams Scholar Award.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard BSMB 781, Oklahoma City, OK 73104, USA. Tel: +1 4052712388; Fax: +1 4052713548; Email: muna-naash{at}ouhsc.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Arikawa, K., Molday, L.L., Molday, R.S. and Williams, D.S. (1992) Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration. J. Cell Biol., 116, 659–667.[Abstract/Free Full Text]

  2. Loewen, C.J. and Molday, R.S. (2000) Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration. J. Biol. Chem., 275, 5370–5378.[Abstract/Free Full Text]

  3. Sanyal, S. and Jansen, H.G. (1981) Absence of receptor outer segments in the retina of rds mutant mice. Neurosci. Lett., 21, 23–26.[CrossRef][Web of Science][Medline]

  4. Sanyal, S. and Zeilmaker, G.H. (1984) Development and degeneration of retina in rds mutant mice: light and electron microscopic observations in experimental chimaeras. Exp. Eye Res., 39, 231–246.[CrossRef][Web of Science][Medline]

  5. Sanyal, S., Dees, C. and Zeilmaker, G.H. (1986) Development and degeneration of retina in rds mutant mice: observations in chimaeras of heterozygous mutant and normal genotype. J. Embryol. Exp. Morphol., 98, 111–121.[Web of Science][Medline]

  6. Hawkins, R.K., Jansen, H.G. and Sanyal, S. (1985) Development and degeneration of retina in rds mutant mice: photoreceptor abnormalities in the heterozygotes. Exp. Eye Res., 41, 701–720.[CrossRef][Web of Science][Medline]

  7. Goldberg, A.F., Moritz, O.L. and Molday, R.S. (1995) Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes. Biochemistry, 34, 14213–14219.[CrossRef][Medline]

  8. Boesze-Battaglia, K. and Goldberg, A.F. (2002) Photoreceptor renewal: a role for peripherin/rds. Int. Rev. Cytol., 217, 183–225.[Web of Science][Medline]

  9. Boesze-Battagliaa, K. and Stefano, F.P. (2002) Peripherin/rds fusogenic function correlates with subunit assembly. Exp. Eye Res., 75, 227–231.[CrossRef][Web of Science][Medline]

  10. Poetsch, A., Molday, L.L. and Molday, R.S. (2001) The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes. J. Biol. Chem., 276, 48009–48016.[Abstract/Free Full Text]

  11. Kohl, S., Giddings, I., Besch, D., Apfelstedt-Sylla, E., Zrenner, E. and Wissinger, B. (1998) The role of the peripherin/RDS gene in retinal dystrophies. Acta Anat. (Basel), 162, 75–84.[CrossRef][Web of Science][Medline]

  12. Wells, J., Wroblewski, J., Keen, J., Inglehearn, C., Jubb, C., Eckstein, A., Jay, M., Arden, G., Bhattacharya, S., Fitzke, F. et al. (1993) Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy. Nat. Genet., 3, 213–218.[CrossRef][Web of Science][Medline]

  13. Wroblewski, J.J., Wells, J.A., III, Eckstein, A., Fitzke, F., Jubb, C., Keen, T.J., Inglehearn, C., Bhattacharya, S., Arden, G.B., Jay, M. et al. (1994) Macular dystrophy associated with mutations at codon 172 in the human retinal degeneration slow gene. Ophthalmology, 101, 12–22.[Web of Science][Medline]

  14. Nakazawa, M., Wada, Y. and Tamai, M. (1995) Macular dystrophy associated with monogenic Arg172Trp mutation of the peripherin/RDS gene in a Japanese family. Retina, 15, 518–523.[Web of Science][Medline]

  15. Jacobson, S.G., Cideciyan, A.V., Maguire, A.M., Bennett, J., Sheffield, V.C. and Stone, E.M. (1996) Preferential rod and cone photoreceptor abnormalities in heterozygotes with point mutations in the RDS gene. Exp. Eye Res., 63, 603–608.[CrossRef][Web of Science][Medline]

  16. Milla, E., Heon, E., Piguet, B., Ducrey, N., Butler, N., Stone, E., Schorderet, D.F. and Munier, F. (1998) [Mutational screening of peripherin/RDS genes, rhodopsin and ROM-1 in 69 index cases with retinitis pigmentosa and other retinal dystrophies]. Klin. Monatsbl. Augenheilkd., 212, 305–308.[Medline]

  17. Payne, A.M., Downes, S.M., Bessant, D.A., Bird, A.C. and Bhattacharya, S.S. (1998) Founder effect, seen in the British population, of the 172 peripherin/RDS mutation and further refinement of genetic positioning of the peripherin/RDS gene. Am. J. Hum. Genet., 62, 192–195.[CrossRef][Web of Science][Medline]

  18. Downes, S.M., Fitzke, F.W., Holder, G.E., Payne, A.M., Bessant, D.A., Bhattacharya, S.S. and Bird, A.C. (1999) Clinical features of codon 172 RDS macular dystrophy: similar phenotype in 12 families. Arch. Ophthalmol., 117, 1373–1383.[Abstract/Free Full Text]

  19. Ekstrom, U., Andreasson, S., Ponjavic, V., Abrahamson, M., Sandgren, O., Nilsson-Ehle, P. and Ehinger, B. (1998) A Swedish family with a mutation in the peripherin/RDS gene (Arg-172-Trp) associated with a progressive retinal degeneration. Ophthalmic Genet., 19, 149–156.[CrossRef][Web of Science][Medline]

  20. Goldberg, A.F., and Molday, R.S. (1996) Defective subunit assembly underlies a digenic form of retinitis pigmentosa linked to mutations in peripherin/rds and Rom-1. Proc. Natl Acad. Sci. USA, 93, 13726–13730.[Abstract/Free Full Text]

  21. Naash, M.I., Hollyfield, J.G., Al Ubaidi, M.R. and Baehr, W. (1993) Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc. Natl Acad. Sci. USA, 90, 5499–5503.[Abstract/Free Full Text]

  22. Kohler, K., Guenther, E. and Zrenner, E. (1997) [Animal models for retinitis pigmentosa research]. Klin. Monatsbl. Augenheilkd., 211, 84–93.[Medline]

  23. Dejneka, N.S., Rex, T.S. and Bennett, J. (2003) Gene therapy and animal models for retinal disease. Dev. Ophthalmol., 37, 188–198.[CrossRef][Medline]

  24. Li, T. (2001) Disease model: photoreceptor degenerations. Trends Mol. Med., 7, 133–135.[CrossRef][Web of Science][Medline]

  25. Cheng, T., Peachey, N.S., Li, S., Goto, Y., Cao, Y. and Naash, M.I. (1997) The effect of peripherin/rds haploinsufficiency on rod and cone photoreceptors. J. Neurosci., 17, 8118–8128.[Abstract/Free Full Text]

  26. Al Ubaidi, M.R., Font, R.L., Quiambao, A.B., Keener, M.J., Liou, G.I., Overbeek, P.A. and Baehr, W. (1992) Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. J. Cell Biol., 119, 1681–1687.[Abstract/Free Full Text]

  27. Liou, G.I., Geng, L., Al Ubaidi, M.R., Matragoon, S., Hanten, G., Baehr, W. and Overbeek, P.A. (1990) Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein. J. Biol. Chem., 265, 8373–8376.[Abstract/Free Full Text]

  28. Yokoyama, T., Liou, G.I., Caldwell, R.B. and Overbeek, P.A. (1992) Photoreceptor-specific activity of the human interphotoreceptor retinoid-binding protein (IRBP) promoter in transgenic mice. Exp. Eye Res., 55, 225–233.[Web of Science][Medline]

  29. Liou, G.I., Matragoon, S., Yang, J., Geng, L., Overbeek, P.A. and Ma, D.P. (1991) Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region. Biochem. Biophys. Res. Commun., 181, 159–165.[CrossRef][Web of Science][Medline]

  30. Fei, Y., Matragoon, S., Smith, S.B., Overbeek, P.A., Chen, S., Zack, D.J. and Liou, G.I. (1999) Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone–rod-homeobox element is essential for photoreceptor-specific expression in vivo. J. Biochem. (Tokyo), 125, 1189–1199.[Abstract/Free Full Text]

  31. Jomary, C., Chatelain, G., Michel, D., Weston, A., Neal, M.J. and Jones, S.E. (1999) Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation. J. Cell Sci., 112 (Pt 10), 1455–1464.[Abstract]

  32. Lin, S.C., Skapek, S.X., Papermaster, D.S., Hankin, M. and Lee, E.Y. (2001) The proliferative and apoptotic activities of E2F1 in the mouse retina. Oncogene, 20, 7073–7084.[CrossRef][Web of Science][Medline]

  33. Peachey, N.S., Quiambao, A.B., Xu, X., Pardue, M.T., Roveri, L., McCall, M.A. and Al Ubaidi, M.R. (2003) Loss of bipolar cells resulting from the expression of bcl-2 directed by the IRBP promoter. Exp. Eye Res., 77, 477–483.[CrossRef][Web of Science][Medline]

  34. Quiambao, A.B., Tan, E., Chang, S., Komori, N., Naash, M.I., Peachey, N.S., Matsumoto, H., Ucker, D.S. and Al Ubaidi, M.R. (2001) Transgenic Bcl-2 expressed in photoreceptor cells confers both death-sparing and death-inducing effects. Exp. Eye Res., 73, 711–721.[CrossRef][Web of Science][Medline]

  35. Ying, S., Fong, S.L., Fong, W.B., Kao, C.W., Converse, R.L. and Kao, W.W. (1998) A CAT reporter construct containing 277 bp GNAT2 promoter and 214 bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice. Curr. Eye Res., 17, 777–782.[CrossRef][Web of Science][Medline]

  36. Molday, R.S., Hicks, D. and Molday, L. (1987) Peripherin. A rim-specific membrane protein of rod outer segment discs. Invest. Ophthalmol. Vis. Sci., 28, 50–61.[Abstract/Free Full Text]

  37. Connell, G., Bascom, R., Molday, L., Reid, D., McInnes, R.R. and Molday, R.S. (1991) Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse. Proc. Natl Acad. Sci. USA, 88, 723–726.[Abstract/Free Full Text]

  38. Nour, M., Ding, X.Q., Stricker, H., Fliesler, S.J. and Naash, M.I. (2004) Modulating expression of peripherin/rds in transgenic mice: critical levels and effect of over-expression. Invest. Ophthalmol. Vis. Sci., 45, 2514–2521.

  39. Li, C., Peachey, N. and Naash, M.I. (1999) Animal models with mutations in the peripherin/rds gene. Invest. Ophthalmol. Vis. Sci., 40, S202.

  40. Goldberg, A.F. and Molday, R.S. (1996) Subunit composition of the peripherin/rds-Rom-1 disk rim complex from rod photoreceptors: hydrodynamic evidence for a tetrameric quaternary structure. Biochemistry, 35, 6144–6149.[CrossRef][Medline]

  41. Moritz, O.L. and Molday, R.S. (1996) Molecular cloning, membrane topology and localization of bovine Rom-1 in rod and cone photoreceptor cells. Invest. Ophthalmol. Vis. Sci., 37, 352–362.[Abstract/Free Full Text]

  42. Goldberg, A.F., Fales, L.M., Hurley, J.B. and Khattree, N. (2001) Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies. J. Biol. Chem., 276, 42700–42706.[Abstract/Free Full Text]

  43. Goldberg, A.F. and Molday, R.S. (2000) Expression and characterization of peripherin/rds-Rom-1 complexes and mutants implicated in retinal degenerative diseases. Method. Enzymol., 316, 671–687.[Web of Science][Medline]

  44. Clarke, G., Goldberg, A.F., Vidgen, D., Collins, L., Ploder, L., Schwarz, L., Molday, L.L., Rossant, J., Szel, A., Molday, R.S., Birch, D.G. and McInnes, R.R. (2000) Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis. Nat. Genet., 25, 67–73.[CrossRef][Web of Science][Medline]

  45. Nakamoto, R.K., Verjovski-Almeida, S., Allen, K.E., Ambesi, A., Rao, R. and Slayman, C.W. (1998) Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis. J. Biol. Chem., 273, 7338–7344.[Abstract/Free Full Text]

  46. Ding, X.Q., Pinon, D.I., Furse, K.E., Lybrand, T.P. and Miller, L.J. (2002) Refinement of the conformation of a critical region of charge–charge interaction between cholecystokinin and its receptor. Mol. Pharmacol., 61, 1041–1052.[Abstract/Free Full Text]

  47. Chang, A., Rose, M.D. and Slayman, C.W. (1993) Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65. Proc. Natl Acad. Sci. USA, 90, 5808–5812.[Abstract/Free Full Text]

  48. Kedzierski, W., Lloyd, M., Birch, D.G., Bok, D. and Travis, G.H. (1997) Generation and analysis of transgenic mice expressing P216L-substituted rds/peripherin in rod photoreceptors. Invest. Ophthalmol. Vis. Sci., 38, 498–509.[Abstract/Free Full Text]

  49. Kedzierski, W., Nusinowitz, S., Birch, D., Clarke, G., McInnes, R.R., Bok, D. and Travis, G.H. (2001) Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa. Proc. Natl. Acad. Sci. USA, 98, 7718–7723.[Abstract/Free Full Text]

  50. McNally, N., Kenna, P.F., Rancourt, D., Ahmed, T., Stitt, A., Colledge, W.H., Lloyd, D.G., Palfi, A., O'Neill, B., Humphries, M.M., Humphries, P. and Farrar, G.J. (2002) Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene. Hum. Mol. Genet., 11, 1005–1016.[Abstract/Free Full Text]

  51. Piguet, B., Heon, E., Munier, F.L., Grounauer, P.A., Niemeyer, G., Butler, N., Schorderet, D.F., Sheffield, V.C. and Stone, E.M. (1996) Full characterization of the maculopathy associated with an Arg-172-Trp mutation in the RDS/peripherin gene. Ophthalmic Genet., 17, 175–186.[Web of Science][Medline]

  52. Mears, A.J., Kondo, M., Swain, P.K., Takada, Y., Bush, R.A., Saunders, T.L., Sieving, P.A. and Swaroop, A. (2001) Nrl is required for rod photoreceptor development. Nat. Genet., 29, 447–452.[CrossRef][Web of Science][Medline]

  53. Tan, E., Wang, Q., Quiambao, A.B., Xu, X., Qtaishat, N.M., Peachey, N.S., Lem, J., Fliesler, S.J., Pepperberg, D.R., Naash, M.I. and Al Ubaidi, M.R. (2001) The relationship between opsin overexpression and photoreceptor degeneration. Invest. Ophthalmol. Vis. Sci., 42, 589–600.[Abstract/Free Full Text]

  54. Chiu, M.I. and Nathans, J. (1994) A sequence upstream of the mouse blue visual pigment gene directs blue cone-specific transgene expression in mouse retinas. Vis. Neurosci., 11, 773–780.[Web of Science][Medline]

  55. Nour, M., Quiambao, A.B., Al Ubaidi, M.R. and Naash, M.I. (2004) Absence of functional and structural abnormalities associated with expression of EGFP in the retina. Invest. Ophthalmol. Vis. Sci., 45, 15–22.[Abstract/Free Full Text]

  56. Nour, M., Quiambao, A.B., Peterson, W.M., Al Ubaidi, M.R. and Naash, M.I. (2003) P2Y(2) receptor agonist INS37217 enhances functional recovery after detachment caused by subretinal injection in normal and rds mice. Invest. Ophthalmol. Vis. Sci., 44, 4505–4514.[Abstract/Free Full Text]

  57. Li, C., Ding, X.Q., O'Brien, J., Al Ubaidi, M.R. and Naash, M.I. (2003) Molecular characterization of the skate peripherin/rds gene: relationship to its orthologues and paralogues. Invest. Ophthalmol. Vis. Sci., 44, 2433–2441.[Abstract/Free Full Text]

  58. Goldberg, A.F., Loewen, C.J. and Molday, R.S. (1998) Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa. Biochemistry, 37, 680–685.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
X.-Q. Ding, C. S. Harry, Y. Umino, A. V. Matveev, S. J. Fliesler, and R. B. Barlow
Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism
Hum. Mol. Genet., December 15, 2009; 18(24): 4770 - 4780.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
D. Chakraborty, X.-Q. Ding, S. M. Conley, S. J. Fliesler, and M. I. Naash
Differential requirements for retinal degeneration slow intermolecular disulfide-linked oligomerization in rods versus cones
Hum. Mol. Genet., March 1, 2009; 18(5): 797 - 808.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Conley, M. Nour, S. J. Fliesler, and M. I. Naash
Late-Onset Cone Photoreceptor Degeneration Induced by R172W Mutation in Rds and Partial Rescue by Gene Supplementation
Invest. Ophthalmol. Vis. Sci., December 1, 2007; 48(12): 5397 - 5407.
[Abstract] [Full Text] [PDF]

Home page
 Br J OphthalmolHome page
C. J F Boon, M. J van Schooneveld, A. I den Hollander, J. J C van Lith-Verhoeven, M. N Zonneveld-Vrieling, T. Theelen, F. P M Cremers, C. B Hoyng, and B J. Klevering
Mutations in the peripherin/RDS gene are an important cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus
Br J Ophthalmol, November 1, 2007; 91(11): 1504 - 1511.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
A. F. X. Goldberg, L. M. Ritter, N. Khattree, N. S. Peachey, R. N. Fariss, L. Dang, M. Yu, and A. R. Bottrell
An Intramembrane Glutamic Acid Governs Peripherin/rds Function for Photoreceptor Disk Morphogenesis
Invest. Ophthalmol. Vis. Sci., July 1, 2007; 48(7): 2975 - 2986.
[Abstract] [Full Text] [PDF]

Home page
 Arch OphthalmolHome page
C. N. Keilhauer, T. Meigen, and B. H. F. Weber
Clinical Findings in a Multigeneration Family With Autosomal Dominant Central Areolar Choroidal Dystrophy Associated With an Arg195Leu Mutation in the Peripherin/RDS Gene
Arch Ophthalmol, July 1, 2006; 124(7): 1020 - 1027.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/18/2075    most recent
ddh211v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (21)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ding, X.-Q.
Right arrow Articles by Naash, M. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ding, X.-Q.
Right arrow Articles by Naash, M. I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?