| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Structural and functional rescue of murine rod photoreceptors by human rhodopsin transgene
Introduction
Results And Discussion
Materials And Methods
Acknowledgements
References
Structural and functional rescue of murine rod photoreceptors by human rhodopsin transgene
Received February 25, 1999; Revised and Accepted April 21, 1999
Mice carrying a targeted disruption of the rhodopsin gene develop a severe degenerative retinopathy, failing to elaborate rod photoreceptor outer segments (ROS), having no recordable rod electroretinogram (ERG) and losing all of their rod cells over a period of ~12 weeks. Murine and human rhodopsins differ in their amino acid sequences. Whether, or to what extent, such variability might influence the ability of human rhodopsin to serve as an adequate structural and functional substitute for the endogenous protein in mouse rod cells bears direct relevance to exploiting the full utility of Rho-/- animals as a model of degenerative retinal disease in man. We crossed Rho-/- mice with mice expressing a wild-type human rhodopsin transgene at levels approximating to those of the endogenous protein. Immunohistological examination of retinal selections from such animals demonstrated ROS of normal number and length and temporal expression of rhodopsin similar to that observed in wild-type animals; that is, immunoreactivity to an anti-rhodopsin antibody became clearly evident by day 3 post-partum. Whereas Rho-/- mice never display a rod ERG response, and even lose cone responses by 12 weeks of age, rescued mice showed 75% normal maximum amplitudes and had ERG b-wave thresholds (based on a 50 µV criterion) within 0.1 log unit of normal wild-type at 20 weeks, and cone amplitudes remained normal at this age. These data demonstrate very substantial structural and functional rescue of the rod photoreceptors of Rho-/- mice and long-term preservation by the human rhodopsin transgene.
INTRODUCTION
The group of conditions collectively termed retinitis pigmentosa (RP) is both clinically and genetically heterogeneous (1,2). Major features of the condition are early nyctalopia (night blindness) owing to dysfunction and death of the rod photoreceptor cells, a symptom usually followed by narrowing of the visual fields due to the subsequent loss of the cone photoreceptors. Intraretinal pigmentary deposits are often seen as the disease progresses (for reviews, see refs 3 and 4). RP currently affects up to 1.5 million people world-wide, in most cases resulting in significant visual handicap. Up to 50% of cases are sporadic, the remainder segregating in an autosomal dominant, recessive or X-linked recessive mode. Approximately 40 genetic loci involved in the aetiology of the disease or of syndromes incorporating the disease have been identified, largely as a result of genetic linkage studies, and up to 15 genes have been identified (http://www.sph.uth.tmc.edu/retnet/ ). The majority of proteins encoded by such genes are expressed within the photoreceptor cells and are components either of the visual transduction cycle or structural components of these cells. The localization of the first gene to be implicated in autosomal dominant RP (adRP) on the long arm of chromosome 3 was reported in 1989 (5,6). The gene encoding rhodopsin, the primary photoreactive pigment of the rod photoreceptor cells, had previously been shown to map within this region of the genome, and a mutation within that gene, P23H, was subsequently identified in a family with RP (7). To date, at least 90 mutations have been identified within the rhodopsin gene, the majority in adRP, although a few mutations implicated in recessive forms of the disease or in congenital night blindness have also been identified (8-12).
Rhodopsin is the most abundantly expressed protein of the rod photoreceptor cells (13). The characterization of a mouse model for degenerative retinal disease, based upon the targeted disruption of the rhodopsin gene, was reported by this team and its collaborators in 1997 (14) and further electroretinographic studies on such animals have been reported recently (15). Rho-/- mice do not elaborate rod photoreceptor outer segments (ROS); the rod electroretinogram (ERG) is unrecordable and the photoreceptor layer is reduced to a single row of nuclei, probably representing those derived exclusively from cone cells, over a period of ~3 months. In order to fully exploit the utility of these animals in the development of methods for the re-introduction to ocular tissues of functional rhodopsin genes in man, or in the development of suppression effectors of dominant mutations within the human rhodopsin gene, it would be of value to assess the ability of human rhodopsin to rescue the retinopathy in such mice.
Murine and human rhodopsins differ in their amino acid sequences at 18 residues. Interestingly, inter-species variability is seen around the chromophore-binding lysine at codon 296, an area that arguably would be expected to be significantly conserved. Whether, or to what extent, such variability has any influence on the ability of human rhodopsin to couple with similar efficiency as that of the endogenous protein to the signal transduction system of the mouse and thus effectively rescue the photoreceptors of Rho-/- mice, or to perform as an adequate structural substitute in the mouse ROS, requires formal establishment. Here we report the results of experiments in which Rho-/- mice were crossed onto a transgenic strain of mouse expressing wild-type human rhodopsin protein at levels which approximate to those normally existing in the mouse retina (16). Histopathological, immunological and electroretinographic data are presented which demonstrate extensive structural and functional rescue of the mouse photoreceptors by the human transgene.
RESULTS AND DISCUSSION
Mice carrying a targeted disruption of the rhodopsin gene show a steady decline of photoreceptor numbers with age. At 90 days, the outer nuclear layer (ONL) is reduced to only a single layer of cells, which are primarily surviving cone photoreceptors. Using immunocytochemical techniques, rhodopsin is undetectable within the degenerating photoreceptor layer at any time, and even young Rho-/- mice have no rod-driven ERG responses (14).
Sections of retinas of 3-month-old Rho+/+ and Rho-/- mice, and of mice expressing the human rhodopsin transgene, are shown in Figure 1. At this age, Rho-/- mice have lost essentially all of their photoreceptors. In terms of numbers of nuclei within the ONL, and of morphology of inner and outer segments of the photoreceptors, wild-type retinas and retinas rescued by the human transgene were indistinguishable.
Figure 1. Epon-embedded sections of the retinas of 3-month-old Rho-/- (A) and wild-type mice (B), and of retinas from Rho-/- mice rescued by human transgene (C).
Immunocytochemical studies were performed on cryo-sectioned retinas using a fluorescent anti-rhodopsin antibody, 4D2, kindly supplied by Drs Robert and Lauri Molday. This antibody recognizes an epitope at the N-terminus of the rhodopsin protein. No immunoreactivity was detectable in retinal sections of Rho-/- mice at any stage of development. No reactivity was detectable in the retinas of wild-type and rescued mice at 1 day of age, but was clearly visible at day 3 (Fig. 2). At 3 months, immunofluorescence was confined exclusively to the ROS in both wild-type and transgene-rescued retinas (Fig. 2). The results obtained from wild-type and rescued retinas were similar.
Figure 2. Immunocytochemical analysis of cryosectioned mouse retinas using fluorescent anti-rhodopsin antibody 4D2. Upper panel (left to right): DIC image of 3-day-old Rho-/- mouse retina; immunoreactivity toward same retinal section; DIC image of 3-day-old wild-type mouse retina; immunoreactivity toward same retinal section; DIC image of 3-day-old Rho-/- retina rescued by human rhodopsin transgene; and immunoreactivity toward same retinal section. Lower panel: as above sequence but with retinas of 3-month-old animals.
Figure 3 shows a typical dark-adapted ERG series recorded over 6.4 log units of flash intensity for the wild-type (left) and the transgenic mice (right). The normal negative a-wave and positive b-wave responses are seen for bright flashes at the bottom of the figure. The dimmest flashes primarily show the positive b-wave and a negativity that is the scotopic threshold response from the proximal retina. For both groups, the ERG threshold of 10 µV is at approximately -5.14 log candela/s/m2 (cd-s/m2). The homozygous Rho-/- mice had no detectable ERG response for any stimulus, including the brightest flash for both dark- and light-adapted conditions.
Figure 3. Typical dark-adapted ERG intensity series from a normal wild-type mouse (left) and a transgenic mouse (right), closest to the means of the respective groups. Amplitude calibration is for adjacent and lower traces for 500 µV, and adjacent and upper traces for 20 µV. The vertical mark on the time scale indicates the stimulus flash.
Figure 4 shows the intensity-response functions for the normal wild-type and the transgenic mice. The wild-type mice have slightly larger b-wave amplitudes than transgenic mice at intensities above -1 log cd-s/m2, but the amplitude difference is not significant. The maximum b-wave amplitude measured at a flash intensity level of 0.61 log cd-s/m2 was 792 488 µV for the wild-type mice and 616 184 µV for the transgenic mice (Table 1; P = 0.52 by a two-tailed student t-test). A 50 µV criterion was used to identify b-wave threshold, and the corresponding flash intensity level was measured from the intensity-response curve for each animal. The mean b-wave threshold for normal mice was -3.99 0.75 log cd-s/m2 and the mean for the transgenic mice was -4.00 0.14 log cd-s/m2, which is not significantly different for the two groups (Table 1; P = 0.93 by a two-tailed t-test).
Figure 4. ERG intensity-response curve of the mean b-wave amplitude for each group, with bars indicating SE. Fifty microvolt threshold criterion is shown by dashed line.
Table 1. Summary of ERG data
| Type | Number | Scotopic thresholdalog cd-s/m2 | Scotopic b-waveV-max (µV) | Photopic b-waveV-max (µV) |
| Wild Type | 4 | -3.99 (0.75 SD) | 792 (488 SD) | 150 (71 SD) |
| Transgenic | 4 | -4.00 (0.14 SD) | 616 (184 SD) | 155 (112 SD) |
| Homozygous | 4 | No detectable ERG responses to the brightest flash | ||
The maximum amplitude of the cone-driven photopic ERG for a 0.61 log cd-s/m2 flash was 150 71 µV for the wild-types and 155 112 µV for the transgenic mice and was not significantly different (P = 0.94 by a two-tailed t-test).
Wild-type animals used in this investigation had rod thresholds quite similar to those reported previously for wild-type mice (15). The same study found that the dark-adapted b-wave threshold of Rho-/- mice was at -1.07 log cd-s/m2 for animals younger than 6 weeks of age; since this represents cone threshold, all Rho-/- responses at dimmer intensities must be rod driven (15). By 12 weeks of age, the retinal degeneration in the Rho-/- mouse resulted in no detectable ERG even with bright flashes. The transgenic animals, however, have thresholds 3.0 log units more sensitive than the cone dark-adapted b-wave threshold of Rho-/- mice in the previous studies (14,15), indicating that the human rhodopsin transgene against a Rho-/- background has rescued rod function. Furthermore, these recordings were made in 20-week-old mice, by which age the Rho-/- animals have no detectable response, both in the current and in the previous studies (14,15).
In summary, the histopathological, immunocytochemical and electrophysiological data presented here demonstrate a substantial structural and functional rescue of rod cells in the Rho-/- mouse retina by crossing onto a transgenic strain of mouse expressing the human rhodopsin gene. This information is required for optimal use of Rho-/- mice in exploring methods for suppressing human degenerative retinal conditions whose molecular pathologies are based either upon loss of rhodopsin gene function or on the manifestation of dominant mutations within that gene.
MATERIALS AND METHODS
Methods for genotypic analysis of Rho-/- mice based on amplification of tail DNAs have been described previously (16). Mice homozygous for the presence of a human rhodopsin transgene were kindly made available by Dr T. Dryja (IMA Eye and Ear Infirmary, Boston, MA)(16). In this line of transgenic mouse, levels of the human opsin approximate to those of the endogenous protein in the wild-type mouse. Primers specific for amplification of exon 5 of the human rhodopsin gene were: sense, 5[prime]-TTCCAAGCACACTGTGGGCA-3[prime]; and antisense, 5[prime]-TGTGACTTCGTTCATTCTGC-3[prime]. A 25 µl PCR reaction mix contained 100 ng DNA, 50 pmol sense and antisense primers, 200 µM each of dGTP, dATP, dTTP and dCTP, 1.5 mM MgCl2, 100 mM Tris, pH 9, 50 mM KCl, 1× Triton and 0.75 U Taq polymerase. PCR conditions were 94°C for 1 min, 55°C for 1 min and 72°C for 1.2 min, for 30 cycles. The amplification product obtained with these primers is 274 bp in length. Rho-/- mice were crossed with mice homozygous for the human rhodopsin transgene. F1s, heterozygous for both the mouse and human rhodopsin genes, were self crossed, and those of genotype Rho-/- and carrying either one or two copies of the human rhodopsin transgene were identified for use in the present study (note that the assay for the human opsin gene does not discriminate between animals heterozygous or homozygous for the human gene).
Methods for histopathological, immunocytochemical and electroretinographic analyses of mouse retinas have been described previously (14).
ACKNOWLEDGEMENTS
The Ocular Genetics Research Unit at Trinity College Dublin is supported by the Wellcome Trust, Fighting Blindness Ireland, the British RP Association, Foundation Fighting Blindness (USA) and the Health Research Board of Ireland. Also supported by ROI-EY06094, P30-EY07003 (Vision Core) and an RPB Senior Scientific Investigator Award (P.A.S.). A.H.H. is a Marie Curie Fellow, funded as part as a European Community training project financed by the Commission under the Training and Mobility of Researchers Programme. Mice carrying the human rhodopsin transgene were kindly provided by Dr T. Dryja (IMA Eye and Ear Infirmary, Boston, MA).
REFERENCES
*To whom correspondence should be addressed. Tel: +353 1 6082484; Fax: +353 1 6719394; Email: gjfarrar{at}vax1.tcd.ie
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification:
Copyright© Oxford University Press, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Deretic, A. H. Williams, N. Ransom, V. Morel, P. A. Hargrave, and A. Arendt Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4) PNAS, March 1, 2005; 102(9): 3301 - 3306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Menon, M. Han, and T. P. Sakmar Rhodopsin: Structural Basis of Molecular Physiology Physiol Rev, October 1, 2001; 81(4): 1659 - 1688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Sieving, M. L. Fowler, R. A. Bush, S. Machida, P. D. Calvert, D. G. Green, C. L. Makino, and C. L. McHenry Constitutive "Light" Adaptation in Rods from G90D Rhodopsin: A Mechanism for Human Congenital Nightblindness without Rod Cell Loss J. Neurosci., August 1, 2001; 21(15): 5449 - 5460. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Durbeej and K. P. Campbell Biochemical Characterization of the Epithelial Dystroglycan Complex J. Biol. Chem., September 10, 1999; 274(37): 26609 - 26616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Imamura and E. Ozawa Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes PNAS, May 26, 1998; 95(11): 6139 - 6144. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||