CARACTERIZACIÓN DE LAS MATERIAS PRIMAS

The rd mouse is a naturally occurring autosomal recessive animal model for retinal degeneration that has been studied in great detail The phenotype involves the degeneration of photoreceptors after the second week of life and virtually all rod cells disappear by postnatal day 20 Although cone receptors survive to this stage, subsequently they begin to degenerate but at a slower rate than the rods. Before the onset of cell degeneration elevated levels of cGMP are detected in the rd retina, which is followed by a steep decline in cGMP levels to barely detectable levels when all photoreceptors have disappeared. The initial rise in cGMP correlates with a deficiency in rod specific cGMP PDE (pdeb) as the rd locus (reviewed by Farber, 1995). The mutation in the rd mouse is recessive owing to a premature stop codon and an insertion of viral DNA in the pdeb gene, which results in no enzyme production (Bowes et a l, 1990, Pittler and Baehr, 1991; Farber 1995). Similarly, a homozygous nonsense mutation identified in the canine homolog of rod cQM?-PDEB was found mutated to cause the rod/cone dysplasia type 1 {rcdl) in Irish setter dogs (Suber et al, 1993). The rd mouse is a good model for a subset of autosomal recessive RP caused by null mutations in the PDEB gene. The increased level of cGMP has now been proposed to trigger photoreceptor death as prior onset of cell degeneration elevated levels of cGMP are detected in the rd retina.

The retinal degeneration slow {rds) mouse is phenotypically characterised by the abnormal development o f retinal photoreceptors followed by their slow degeneration without any o f the other cell types of the retina being affected (Van Nie et a l, 1978).

This is also a naturally occurring animal model. In rds/rds homozygotes the retina undergoes normal development and differentiation until the first postnatal week when the photoreceptors normally appear. While the other retinal cells continue their normal development, rds/rds fail to form outer segment discs even though the inner segments, including the ciliary projections, appear morphologically normal (Sanyal 1987, Cohen 1983). The process of photoreceptor degeneration escalates then become s more gradual with significantly reduced thickness of the outer nuclear layer; by one year o f age the degeneration is complete (Sanyal 1987). The defect in rds/rds mouse is a pure structural defect as all the components of the visual cascade are present even if at greatly reduced levels (Cohen 1983; Reuter and Sanyal 1984). The rds mutation is not recessive as originally thought since the rds!+ heterozygote mice also exhibit mild phenotype abnormality. In contrast to homozygotes, heterozygotes do form outer segments, which are reduced in length and contain irregularly arranged discs that appear swollen and vacuolated with very slow degeneration (Sanyal, 1987). The phenotype observed in the rds mouse is caused by an insertion mutation that disrupts the gene encoding the rds/peripherin, which produces a null allele (Travis et a l, 1989, 1991). Peripherin is a photoreceptor specific transmembrane protein that is expressed in the rim region of the outer segment discs of both rods and cones. The phenotype of the rds/rds mice show peripherin is essential for the biogenesis o f photoreceptor outer segments. Due to the phenotype in rds mice, the production of aberrant disc structure has been proposed as means of triggering photoreceptor degeneration.

Humphries and co-workers have recently generated mice carrying a targeted disruption of the rhodopsin gene {Rho-i-). The mice do not develop rod outer segments or develop any ERG response (electroretinograph) response after 8 weeks but lose their photoreceptors over 3 months. The heterozygous Rho+/- mice retain majority o f their photoreceptors although the inner and outer segments o f these cells display some structural disorganisation, the outer segment becoming shorter in older mice. Therefore, the rho knockout mouse appears as a good animal model for autosomal recessive RP caused by null mutations in the rhodopsin gene.

The rho mice can also be used for innumerable experiments, which include somatic gene therapy to see if the null phenotype can be rescued using one of the

recombinant viral delivery systems. Success o f such experiments will pave for similar therapeutic intervention in the cognate human disease. Other experiments relate to the creation o f transgenic mice that mimic human dominant RP disease. Previously when transgenic mice were created to develop theories concerning the pathological processes induced by certain rhodopsin, such as Pro23His (Roof et a l, 1994), Gln344Ter (Sung et ah, 1994) and Pro347Ser (Li et al., 1996) by necessity the mutant transgenes were placed on a wild type genetic background. Now these transgenes can be placed in the rho+/- mice background to generate more faithful animal models of dominant RP since humans with Rhodopsin mediated dominant RP are also hemizygous for the wild type allele. Moreover the knockout mouse presents an opportunity to study the effects of mutations especially those mutations that affect the post-translational modification o f rhodopsin, without the confounding presence of wild-type rhodopsin. Such an experiment carried out in vivo might also lead to the recognition o f other proteins that interact with rhodopsin for the correct folding o f rhodopsin which by virtue of their function be candidate genes for RP. Furthermore, experiments carried out on the null background can clarify some post-translational anomalies observed with certain rhodopsin mutations in different experimental systems.

1.9.7.1 Apoptosis

The mouse models {rd, rds and rhodopsin transgenic mice [either Pro347Ser or Gln344ter]) have been used in two independent studies to understand the pathway leading from the primary defect (i.e. mutation in gene) to photoreceptor cell death (Chang et a l, 1993; Portera-Calliau et al., 1994). It was observed that even though the animal models represented different basic mutations, subsequent cell death was remarkably similar and bore all the biochemical hallmarks o f death by apoptosis such as cytoplasmic condensation, nuclear chromatin condensation and inter-nucleosomal DNA fragmentation seen in agarose gels. Cleavage o f DNA that link multiple nucleosomes gives rise to a DNA ladder, composed of fragments that are multiples of 180-200 bp and diagnostic for cell death by apoptosis. Apoptotic cell death is distinct from cell death by necrosis or accidental cell death that produce a spectrum of DNA fragment sizes without

evidence of a nucleosomal ladder (Collins et al., 1992). Apotosis is normally used by retinal cells during development to fine-tune the number of cells in the retina and their interconnections (Young, 1984).

Even though apoptosis has been recognised as the final pathway the mechanisms by which each mutation trigger apoptosis is still not fully understood. However some aspects of the effects of these different mutations suggests certain possibilities. Intemucleosomal DNA fragmentation, a hallmark of apoptotic cell death, is thought to be mediated by a nuclear endonuclease that can be stimulated by a rise in Ca^^ (McConkey et al., 1989). Such a rise in Ca^^ can be induced in the rd mouse where cGMP accumulates as a result of the mutation (Yau and Baylor, 1989), thus activating the endonuclease. In r^A/peripherin mice, failure to develop an outer segment may upset some internal mechanism of the photoreceptor thus activating the endonuclease. This situation is similar to that of a cell under stress, which might cause the cell to self- destruct. In the case of rhodopsin mutations (e.g. Pro347Ser) it could be that the presence of the mutant rhodopsin alters the normal cellular pathways and disrupts normal cell-cell interactions (Huang et a l, 1993), leading to the transmission of an incorrect signal to the photoreceptors and thus causing them to self-destruct.