Mechanism of non-autonomous death in photoreceptor degeneration mutants

Many diseases that ultimately lead to blindness are caused by the degeneration of photoreceptor cells, the rods and cones. Non-autonomous photoreceptor death is caused when a disease gene is expressed in e.g. rods only, but cones also die. The spread of non-autonomous death is the cause of blindness in retinitis pigmentosa (RP), and likely other, very common diseases in humans, including age related macular degeneration (AMD) (Retnet, http://www.sph.uth.tmc.edu; Clin Genet, 2000. 57(5): p. 313-29). These diseases are late-onset, and there is early diagnosis of the degeneration, making it possible to intervene before vision is lost. Moreover, if we knew how to arrest photoreceptor degeneration, there is an excellent possibility for gene therapy through infection of the adjacent retinal pigmental epithelium (RPE) with viral vectors. The RPE has been shown to stably express genes transduced by adeno-associated vectors. The most striking example is a treated Briard dog, Lancelot, who is still showing therapeutic effects for his inherited blindness 3 years following infection (Nat Genet. 2001, 28:92-5). However, we are missing a key piece of information for this type of therapy: the identity of a factor(s) that could be delivered to keep the photoreceptors alive for most cases of human blindness. This is the goal of our studies.

We are using retinal microarrays to define the gene expression changes that accompany photoreceptor death in mouse genetic models of non-autonomous photoreceptor death, with an emphasis on the events that lead to cone death (Figure 1). Cones are a primary target as these are the photoreceptor cells that allow high acuity and daylight vision, while rods are used only in very dim light. We are establishing methods that allow for the initiation of photoreceptor death in a focal manner in various genetic backgrounds. This will allow us to follow the course of non-autonomous death through a retina over time, examining the retina for cellular and molecular changes during the spread of death. In addition, we have discovered an interesting model in which the spread of death is arrested in the cyclin D1 mutant mouse (Figure 2) (Proc Natl Acad Sci U S A.1998, 95:9938-43). This model will be investigated for gene expression changes that accompany the arrest of the spread of death. In all cases, we are using in situ hybridization to further characterize the expression patterns of genes that change in their expression level during the spread of death in both normal and pathological tissue. Finally, we will explore the function of some of these genes using genetic approaches in mice.

Figure 1. The relative rates of rod and cone death in progressive degenerations are shown in an idealized fashion. We are currently measuring these rates for 4 models of retinal degeneration in the mouse. Probes for microarrays are being prepared from the retina and pigemented epithelium at several time points. By comparing across models, we hope to gain some insight into the molecular events that lead to the onset and progression of the non-autonomous cone death.

Figure 2. The cyclin D1-/- murine retina exhibits a unique form of retinal degeneration. Beginning at approximately postnatal day 6, a few foci of apoptotic photoreceptor cells begin to appear. The death spreads from these first foci, ultimately resulting in "holes" in the outer nuclear layer. However, unlike all other models of retinal degeneration, the cyclinD1 model shows an arrest of the spread of apoptosis, such that the holes reach a stable maximum size by approximately 3 weeks of age. (A) a dark field image of a retina dissected from a postnatal day 180 cyclinD1-/-. The holes are primarily in the central retina. (B) scanning electron micrograph of a P21 cyclin D1-/- retina;  arrows depict 3 different sizes of holes. Scale bar equals 20 microns. (C) cryosection through a P21 wild type retina stained with anti-recoverin (in red; with a nuclear stain shown in green), which is expressed in photoreceptors. (D) a comparable section to that shown in (C), but from a cyclinD1 -/- retina. Note that the mutant retina overall is much smaller, and that the outer nuclear layer is disrupted in the area of a hole (arrowhead). Scale bar is 50 microns for C and D. ONL=outer nuclear layer, INL=inner nuclear layer, OPL=outer plexiform layer, IPL=inner plexiform layer, and GCL=ganglion cell layer.