Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration

University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 01/2011; 52(1):190-8. DOI: 10.1167/iovs.10-5194
Source: PubMed


Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated.
The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy.
The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age.
The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.

Download full-text


Available from: Michael H Farkas,
    • " . , 2008 ; Rio Frio et al . , 2008 ; Dell ' Angelica , 2009 ; Taniguchi - Ikeda et al . , 2011 ; Venturini et al . , 2012 ) . Morphological changes in the RPE of these mutant mice were of particular interest ; where we observed the loss of basal infoldings , the formation of basal deposits beneath the RPE and vacuolization in the cytoplasm ( Graziotto et al . , 2011 ) ."
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in the ubiquitously expressed pre-mRNA processing factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31) cause nonsyndromic dominant retinitis pigmentosa in humans, an inherited retinal degeneration. It is unclear what mechanisms, or which cell types of the retina, are affected. Transgenic mice with the human mutations in these genes display late-onset morphological changes in the retinal pigment epithelium (RPE). To determine whether the observed morphological changes are preceded by abnormal RPE function, we investigated its phagocytic function in Prpf3(T494M/T494M), Prpf8(H2309P/H2309P), and Prpf31(+/-) mice. We observe decreased phagocytosis in primary RPE cultures from mutant mice, and this is replicated by shRNA-mediated knockdown of PRPF31 in human ARPE-19 cells. The diurnal rhythmicity of phagocytosis is almost lost, indicated by the marked attenuation of the phagocytic burst 2 hours after light onset. The strength of adhesion between RPE apical microvilli and photoreceptor outer segments also declined during peak adhesion in all mutants. In all models, at least one of the receptors involved in binding and internalization of shed photoreceptor outer segments was subjected to changes in localization. Although the mechanism underlying these changes in RPE function is yet to be elucidated, these data are consistent with the mouse RPE being the primary cell affected by mutations in the RNA splicing factors, and these changes occur at an early age.
    American Journal Of Pathology 08/2014; 184(10). DOI:10.1016/j.ajpath.2014.06.026 · 4.59 Impact Factor
  • Source
    • "Retinal development is an ideal model system that is tightly controlled through a variety of regulatory mechanisms such as transcriptional regulation, alternative splicing, and microRNA (miRNA) regulation. Dysregulation of any of these processes can lead to retinal disease [1], [2], [3]. To understand the molecular basis of retinal development and diseases, one traditional approach is to identify individual genes responsible for either retinal diseases or the developmental process. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding gene regulation is crucial to dissect the molecular basis of human development and disease. Previous studies on transcription regulatory networks often focused on their static properties. Here we used retinal development as a model system to investigate the dynamics of regulatory networks that are comprised of transcription factors, microRNAs and other protein-coding genes. We found that the active sub-networks are topologically different at early and late stages of retinal development. At early stages, the active sub-networks tend to be highly connected, while at late stages, the active sub-networks are more organized in modular structures. Interestingly, network motif usage at early and late stages is also distinct. For example, network motifs containing reciprocal feedback regulatory relationships between two regulators are overrepresented in early developmental stages. Additionally, our analysis of regulatory network dynamics revealed a natural turning point at which the regulatory network undergoes drastic topological changes. Taken together, this work demonstrates that adding a dynamic dimension to network analysis can provide new insights into retinal development, and we suggest the same approach would likely be useful for the analysis of other developing tissues.
    PLoS ONE 10/2012; 7(10):e46521. DOI:10.1371/journal.pone.0046521 · 3.23 Impact Factor
  • Source
    • "PRPF31 is ubiquitously expressed and sufficient levels are crucial for organ maintenance. Consistently, homozygous Prpf31 knock-out mice are embryonic lethal [33], whereas heterozygous Prpf31 knock-out mice surprisingly display degeneration of the retinal pigment epithelium (RPE) [8]. Thus, different PRPF31 thresholds either lead to degeneration or lethality. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.
    Molecular Neurodegeneration 07/2011; 6(1):56. DOI:10.1186/1750-1326-6-56 · 6.56 Impact Factor
Show more