Transgenic Bcl-2 expressed in photoreceptor cells confers both death-sparing and death-inducing effects.

To examine its potential role within the retina as a modulator of cell death and photoreceptor degeneration, bcl-2 expression was targeted to the photoreceptors of transgenic mice by the human IRBP promoter. Three transgenic families were established, with levels of transgene expression between 0.2 and two-fold relative to that of endogenous bcl-2. The effect of bcl-2 expression on genetically programmed photoreceptor degeneration was evaluated by crossing these transgenic mice with mice that develop a rapid degeneration of rod photoreceptors due to expression of a distinct transgene, SV40 T antigen (Tag). Transgenic Bcl-2 was localized to photoreceptor inner segments and was capable of abrogating the activation of caspase activity and the resulting cell death associated with ectopic expression of Tag. However, Bcl-2 itself ultimately caused photoreceptor cell death and retinal degeneration. Several proteins not expressed normally in Tag or other transgenic retinas undergoing photoreceptor degeneration were induced in the Bcl-2 transgenic retinas. Analysis by mass spectroscopy identified one of these proteins as alphaA-crystallin, a member of a protein family that associates with cellular stress. Since Bcl-2 can promote as well as spare cell death in the same photoreceptor population, its potential utility in ameliorating photoreceptor death in human hereditary blinding disorders is compromised.

The relationship between opsin overexpression and photoreceptor degeneration.

PURPOSE: To characterize the process by which overexpression of normal opsin leads to photoreceptor degeneration. METHODS: Three transgenic mouse lines were generated that express different levels of an opsin with three amino acid modifications at the C terminus. These modifications created an epitopic site that can be readily distinguished from the endogenous protein using a bovine opsin-specific antibody. Evidence of degeneration associated with opsin overexpression was provided by anatomic studies and electroretinogram (ERG) recordings. Western blot analysis was used to confirm the production of the transgenic opsin, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of opsin overexpressed in each line. Immunocytochemistry was used to determine the cellular localization of transgenic opsin. Amounts of 11-cis retinal were determined by extraction and high-performance liquid chromatography (HPLC). RESULTS: Opsin expression levels in the three lines were found to be 123%, 169%, and 222% of the level measured in nontransgenic animals, providing direct correlation between the level of transgene expression and the severity of the degenerative phenotype. In the lower expressing lines, ERG a-wave amplitudes were reduced to less than approximately 30% and 15% of normal values, whereas responses of the highest expressing line were indistinguishable from noise. In the lowest expressor, a 26% elevation in 11-cis retinal was observed, whereas in the medium and the high expressors, 11-cis retinal levels were increased by only 30% to 33%, well below the 69% and 122% increases in opsin levels. CONCLUSIONS: The overexpression of normal opsin induces photoreceptor degeneration that is similar to that seen in many mouse models of retinitis pigmentosa. This degeneration can be induced by opsin levels that exceed by only approximately 23% that of the normal mouse retina. Opsin overexpression has potential implications in retinitis pigmentosa.

Degeneration of cone photoreceptors induced by expression of the Mas1 protooncogene.

Although transgenic expression of oncogenes typically leads to tumorigenesis, oncogene expression directed to the rod photoreceptors leads to cell death without tumor formation. To evaluate the cellular and functional changes induced in cone photoreceptors by an oncogene, the Mas1 protooncogene was targeted to the cones of transgenic mice by the human red/green opsin promoter. Mas1 was chosen because of its exclusive expression in the nervous system and its homology to opsin. The overall histologic appearance of the transgenic retina was normal and retinal tumors were never observed. While rod-mediated electroretinograms were normal in all respects, cone-mediated responses were diminished in direct relationship to the level of transgene expression as determined by Northern blot analysis. Responses of UV- and green-sensitive cones were reduced equivalently, and Northern analysis and immunocytochemistry indicated that cone photoreceptor densities were markedly diminished throughout transgenic retinas. These results indicate that oncogene expression in cones induces cell death without tumor formation and support the possibility that aberrant oncogene expression may underlie some forms of hereditary retinal diseases. The Mas1 transgenic mice may be useful in understanding the cone photoreceptor degeneration that occurs in cone dystrophies and age-related macular degeneration and in evaluating potential therapies for these disorders.

A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, approximately 50% of photoreceptors remained and an approximately 45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

Functional consequences of oncogene-induced photoreceptor degeneration in transgenic mice.

This study evaluated retinal function in mice following the expression of oncogenes under the control of photoreceptor-specific promoters in transgenic mice. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit rod- or cone-mediated components. In one transgenic line (MOT1), the simian virus 40 large tumor antigen was expressed under the control of the mouse opsin promoter. MOT1 mice exhibited an age-related decline in the amplitude of the rod-mediated ERG a-wave. In comparison, cone-mediated responses recorded from MOT1 mice remained normal up to four months of age, the oldest age tested. In the second transgenic line (CMYC), the rat c-myc gene was expressed under control of the human interphotoreceptor-retinoid binding protein promoter. CMYC mice exhibited a rapid reduction of cone-mediated responses and a gradual loss of the rod ERG a-wave. Analysis of rod ERG a-waves obtained from young MOT1 and CMYC mice indicated that the rod ERG abnormalities reflect a reduction in the number of rods contributing to the response with the retention of normal response properties in rods that remain. These results support the possibility that aberrant expression of oncogenes may underlie some forms of human rod and cone-rod dystrophy.

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.

We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen.