C. Grimm

Molecular ophthalmology: an update on animal models for retinal degenerations and dystrophies.

For several decades, basic research on acquired and inherited retinal degeneration was substantially based on a variety of animal models, most of them originating from spontaneous mutations, others induced by damaging external agents. In the past few years, however, progress in genetic engineering has led to a rapidly growing number of transgenic animals, mostly mice, carrying constructs that lead to disruption or overexpression of candidate genes for retinal degenerations. On the one hand, these new models constitute a powerful and adaptable tool to investigate the role of specific gene mutations and the resulting cellular defects that finally lead to photoreceptor cell death. On the other hand, they extend the spectrum of animal models suitable for the newly arisen field of retinal somatic gene therapy. To assist researchers and clinicians interested in the field, this article attempts to provide a structured overview on recently developed transgenic animal models as well as on models based on spontaneous mutations and induced degenerations. In this review the authors focus on animal models for photoreceptor degeneration since the rapidly growing field of models for ganglion cell death merits its own review and would be beyond the scope of this article. Even with this restriction, the abundance of information generated especially in the past few years makes the attempt of a complete overview almost illusory. Therefore, we apologise for omissions or shortfalls extant in this review. Furthermore, we want to point out that some of the model systems described have already been used extensively. We will therefore occasionally not cite original publications but rather reviews dealing with the specific model system. Finally, we did not incorporate strategies using viral vectors and/or pharmacological substances. The specific deletion or overexpression of genes susceptible for the modulation of photoreceptor apoptosis, however, was included. The authors are aware that these …

Why study rod cell death in retinal degenerations and how

Age-related macular degeneration (AMD) is a main causes of severe visual impairment in the elderly in industrialized countries. The pathogenesis of this complex diseases is largely unknown, even though clinical characteristics and histopathology are well described. Because several aging changes are identical to those observed in AMD, there appears to exist an unknown switch mechanism from normal ageing to disease. Recent anatomical studies using elegant innovative techniques reveal that there is a 30% rod loss in normal ageing, which is increased in early AMD. Those and other observations by Curcio and co-workers indicate that early rod loss is an important denominator of AMD (Curcio CA. Eye 2001; 15:376). As in retinitis pigmentosa (RP), rods appear to die by apoptosis. Thus it seems mandatory to study the regulation of rod cell death in animal models to unravel possible mechanisms of rod loss in AMD. Our laboratory investigates signal transduction pathways and gene regulation of rod death in our model of light-induced apoptosis. The transcription factor AP1 is essential, whereas other classical pro- and antiapoptotic genes appear to be less important in our model system. Caspase-1 gene expression is distinctly upregulated after light exposure and there are several factors which completely protect against light-induced cell death, such as the anesthetic halothane, dexamethasone and the absence of bleachable rhodopsin during light exposure. A fast rhodopsin regeneration rate increased damage susceptibility. Our data indicate that rhodopsin is essential for the initiation of light-induced rod loss. Following photon absorption, there may be the generation of photochemically active molecules wich then induce the apoptotic death cascade.

Retinal photoreceptors are apoptosis-competent in the absence of JunD/AP-1.

In the retina, apoptosis is an essential component in retinal development and differentiation, but in addition it is also a major component in human retinal degeneration. In many animal models for this human disease group, apoptosis is the final common pathway of photoreceptor cell death. To date, little is known about the molecular mechanisms leading to apoptosis in the retina. Recently, we identified the AP-1 component c-Fos as essential for light-induced photoreceptor apoptosis: non-functional c-Fos completely inhibits lightinduced photoreceptor degeneration in the mouse retina in vivo. To understand the mechanism of this protection it is crucial to learn more about the role of AP-1 and its constituents in the retina. AP-1 is a transcription factor complex that may be differentially assembled from members of the Fos (c-Fos, FosB, Fra-1, Fra-2) and Jun (c-Jun, JunD, JunB) family of proteins. The complex composition of AP-1 determines its biological function by modulating the transcription rate of specific sets of target genes, AP-1 complexes containing cFos exhibiting the strongest transactivation activity. Activation of AP-1 is involved in apoptotic cell death in a variety of different systems. ± 7 We have shown recently that in the mouse retina AP-1 complexes mainly consist of c-Fos, JunD and c-Jun. Interestingly, elevated expression of AP-1 consisting of c-Fos/JunD is also observed during apoptosis in other tissues. To address the role of Jun proteins in AP-1 complexes during photoreceptor apoptosis, we have used transgenic mice lacking functional JunD in our model of light-induced photoreceptor degeneration. The effect of c-Jun depletion could not be tested since cJun knockouts die at midgestation E12.5. JunD mutants (Thepot et al, submitted) were bred from heterozygous matings on a mixed C57BL/6xSv129 background (verification of the genotype by PCR analysis) and maintained in a 12 : 12 h light-dark cycle (lights on at 0600 h) with 10 ± 20 lux within the cages. Six to ten-weeksold junD and junD mice were dark adapted for 16 h and the pupils were dilated with cyclopentolate 1.0% (Alcon Pharmaceuticals, Cham, Switzerland) and phenylephrin 5% (Ciba Vision, Niederwangen, Switzerland). Mice were anaesthetized with CO2 and killed by cervical dislocation either before light exposure (dark control) or after 24 h of darkness following exposure to 15 000 lux of diffuse, cool, white fluorescent light for 2 h. Eyes were removed and processed for light microscopy, in situ analysis of DNA strand breaks (TUNEL) or analysis of genomic DNA by gel electrophoresis. Dark adapted junD and junD mice displayed regular retinal morphology (Figure 1A, a,b) suggesting that physiological apoptosis during development occurred in the absence of JunD. After exposure to 15 000 lux for 2 h and a post-exposure time of 24 h in darkness, both junD and junD mice showed distinct signs of photoreceptor apoptosis (Figure 1A, c,d). Light microscopy revealed condensed photoreceptor nuclei in the outer nuclear layer (ONL), condensed rod inner segments (RIS) and disrupted rod outer segments (ROS). TUNEL staining showed comparable positive labeling of nuclei in the ONL of both groups of animals at this time point (Figure 1B, c,d), whereas the retinas of unexposed junD and junD animals were devoid of signals (Figure 1B, a,b). Similarly, DNA fragmentation was observed in light-exposed junD and junD animals while the DNA of unexposed controls was intact (Figure 1C). These data indicate that photoreceptor apoptosis is independent of functional JunD. In the rodent retina, little is known about the specific role of the AP-1 members c-Fos and JunD during the maintenance of retinal function and morphology. Basal cfos expression follows a diurnal rhythm and is predominantly found in the ONL. Furthermore, elevated levels of c-fos mRNA in the ONL precede photoreceptor apoptosis in light-induced retinal degeneration and in several animal models for inherited retinal dystrophy such as the retinal degeneration (rd) and the retinal degeneration slow (rds) mouse. In contrast, the distribution pattern of JunD remains unchanged in the normal and the rd mouse with high constitutive levels of JunD in both the Inner Nuclear Layer (INL) and ONL. Similarly, the low-level expression of cJun in the INL and ONL of the normal mouse retina remains unchanged in the rd mouse. In conclusion, in this communication we report that JunD is not essential for light-induced apoptosis of photoreceptor cells in the adult mouse retina. Our data further suggest that developmental apoptosis is not affected by the lack of JunD either. Since AP-1 in the mouse retina predominantly consists of c-Fos, c-Jun and JunD, it is conceivable that, in contrast to c-Fos, the lack of JunD is compensated by other Jun family members. Further studies on other transgenic mice such as an inducible c-Jun mutant on a JunD depleted background are needed to test this hypothesis.

Increased light damage susceptibility at night does not correlate with RPE65 levels and rhodopsin regeneration in rats

The susceptibility of rats to light-induced retinal degeneration is increased at night. In mice, an important determinant of light damage susceptibility is the efficacy of rhodopsin regeneration after bleaching. The rate of rhodopsin regeneration is at least partly controlled by RPE65, a protein expressed in the retinal pigment epithelium. We therefore tested a potential involvement of RPE65 and rhodopsin regeneration in the increased light damage susceptibility of rats at night. For this purpose, rats were exposed to visible light at noon or at midnight and extent of light damage was determined by retinal morphology and TUNEL staining. Rpe65 gene expression was analyzed by semiquantitative RT-PCR and levels of RPE65 protein were determined by Western blotting. Rhodopsin regeneration kinetics was determined by measuring rhodopsin content immediately after a strong bleach and after different times of recovery in darkness. Rats were more susceptible to light damage at night as described by Organisciak and collegues [Invest. Ophthalmol. Vis. Sci. 41 (2000) 3694]. Rpe65 gene expression followed a day-night rhythm with highest steady-state mRNA levels at the beginning and lowest levels at the end of the day period. However, RPE65 protein levels remained constant. Rhodopsin regeneration kinetics did not differ during day and night. We conclude that levels of RPE65 protein and rhodopsin regeneration kinetics do not correlate with the increased light damage susceptibility observed in rats at night. Additional genetic or physiologic modifiers may exist in rats that regulate the retinal responsiveness to acute light exposure.

Photostasis and Beyond

Both disc shedding, the degradative part of the renewal cycle of photoreceptor outer segments, and autophagy, the degradation of cellular organelles in inner segments, follow a circadian rhythm in mammals (LaVail, 1976; Reme et al., 1985). The light history of a given animal (Penn and Williams, 1986), however, defines the amplitude of that rhythm and the overall “sensitivity” of the system to light exposure (Reme et al., 1991). Furthermore, a gradual increase of the light signal simulating a natural twilight transition (Terman, in press) can trigger disc shedding at very low light levels (Bush et al., 1990), raise the upper limit of entrainment to light-dark cycles in hamsters (Boulos et al., 1996), and entrain human circadian rhythms (Wirz-Justice et al., in press).

UV- und Lichtschäden des Auges

Kaum eine Feststellung ist einleuchtender als diese: Licht ist notwendig fur alles Leben auf der Erde. Fur das Auge bedeutet Lichtexposition die Erzeugung des Sehreizes, also die Wahrnehmung unserer Welt. Licht kann jedoch bestimmte Strukturen und Funktionen des Auges verandern, ja, es kann sogar irreversible Schaden im Auge hervorrufen. Diese Beobachtung ist -anekdotische Berichte eingeschlossen — seit dem Altertum bekannt. Neu ist jedoch die systematische Erforschung von Lichtschaden und deren Verwendung in Laboratoriumsuntersuchungen als Modell zur molekularen Analyse von dystrophischen (hereditaren) und degenerativen (induzierten) Augenerkrankungen. Sind also Lichtschaden des Auges relevant fur die dermatologische Praxis?