The results presented above provide several important insights into the pathogenesis of RNA splicing factor RP. First, all three mouse models manifest degenerative changes in the RPE. The finding of a similar phenotype in all three models suggests that the RPE is the primary retinal cell type affected in these forms of RNA splicing factor RP, which in turn leads to photoreceptor degeneration over the long term. The RPE changes are associated with decreased photoreceptor function in the Prpf3
-T494M mice, although overt photoreceptor degeneration was not observed in any of the models. The later-onset phenotype in these three mouse models is consistent with the adult-onset phenotype and vision loss observed in many patients with RNA splicing factor forms of RP.7,47,48
These three mouse models provide a platform for future comparative studies to elucidate the mechanism of pathogenesis of the RNA splicing factor forms of RP.
An interesting finding from this work is that all the Prpf
mutant mice develop loss of RPE basal infoldings and sub-RPE deposits. The deposits share features with the basal deposits associated with macular degeneration, including membranous debris and vesicular structures.49
Both the heterozygous and homozygous mice accumulate these deposits, in agreement with the dominant inheritance of the human conditions. Ultrastructural analysis of the retina from one patient with RNA splicing factor RP has been reported. In this sample, from a patient with a mutation in PRPF8
, the RPE was clearly abnormal with loss of basal infoldings, but since the patient had end-stage RP it is difficult to determine whether the RPE abnormalities were a cause or consequence of photoreceptor loss.50,51
While other forms of RP are not known to develop sub-RPE deposits, the RPE and photoreceptors have an intimate relationship and there are several types of retinal degeneration caused by mutations in genes required for RPE function. Some examples are LCA caused by mutations in LRAT
and RP caused by MERTK
Further, RPE phagocytic dysfunction in β5 integrin-deficient mice leads to photoreceptor dysfunction, and accumulation of lipofuscin in RPE cells.56
The RPE is also a major part of the blood-retinal barrier, and therefore regulates the extracellular environment of photoreceptors. One recent study reported that mice which lack the RPE monocarboxylic acid transporter 3 have an altered pH in the sub-retinal space, and that this leads to altered photoreceptor function.57
One possible explanation for the decreased a-wave ERG observed in the older Prpf3
-T494M mice is that the RPE defects observed lead to a similar alteration in the extracellular milieu of photoreceptor outer segments. In short, the RPE and the retina are integrally related, and problems in one can have consequences for the other. Additional studies of the effects of mutations in the three RNA splicing factors on RPE function and its relationship to photoreceptor degeneration are warranted.
The homozygous Prpf3
-T494M and Prpf8
-H2309P knockin mouse lines are viable, demonstrating that the Prpf3
-T494M and Prpf8
-H2309P mutations do not create null alleles, or the animals would have died embryonically, as was recently reported for Prpf3
knockout animals, Prpf8
knockout animals, and Prpf31
knockin and knockout animals (Deramaudt BM, et al. IOVS
2005;46:ARVO E-Abstract 5263).24,33
The use of gene-targeted knockin mice for these studies provided several important advantages over other methods that have been used to study the RNA splicing factor forms of RP to date,44,58
including the ability to study the Prpf3-T494M protein and the Prpf8-H2309P protein in the absence of wild-type Prpf3 or Prpf8 in vivo, which cannot be readily accomplished in cell culture. We have observed that in neither case do the expression levels of the mutant Prpf3
transcripts change, nor does the size of the transcript, ruling out the possibility of the mutation altering a splice signal within the Prpf3
transcripts themselves. Furthermore, the levels and location of Prpf3-T494M protein within the retina are normal, and no nuclear aggregates were seen, in contrast to a recent study in which aggregation of mutant PRPF3 was seen after over-expression of the protein.44
These findings are also consistent with the hypothesis that mutations in these genes produce disease via a dominant mechanism (dominant-negative or gain-of-function), rather than haploinsufficiency.
The location of the Prpf3 and Sm proteins in the periphery of the nuclei of photoreceptor cells is distinct from the typical staining pattern for splicing proteins, which localize to speckles and Cajal bodies throughout the nucleus but exclude nucleoli. The location of Prpf3 and Sm proteins in the mouse is still speckled but peripheral, consistent with recent findings that nocturnal mammal photoreceptor nuclei have a unique inverted pattern with a heterochromatin center surrounded by euchromatin, nascent transcripts as well as the splicing machinery.59
However, the mutant Prpf3-T494M location does not differ from that of the wild-type, implying no nuclear import defects or defects incorporating into the snRNPs.
The findings described here provide a new model for the pathogenesis of RNA splicing RP. Although the characteristic loss of photoreceptors is the defining feature of RP, our mouse models suggest that photoreceptor dysfunction in RNA splicing factor RP may arise secondary to an unhealthy RPE. Given these findings, we hypothesize that production of an aberrantly spliced transcript or group of transcripts in RPE and/or photoreceptor cells is responsible for the retinal degeneration phenotype observed in patients with RNA splicing factor RP. We believe analyses of the transcriptomes of the RPE and retinas of the Prpf3
-H2309P, and Prpf31
-knockout mice will help identify the pathogenic splice alterations responsible for retinal disease. This is a worthwhile endeavor, because identification of the altered transcripts that cause retinal degeneration will open the path to the development of therapies for these blinding disorders. Indeed, there are now several promising examples of the use of antisense oligonucleotides to correct splicing errors caused by mutations in vivo, including a recent report of efficacy in a small clinical trial.60,61