Purpose Previous studies suggest that ceramide is a proapoptotic lipid as high levels of ceramides can lead to apoptosis of neuronal cells, including photoreceptors. However, no pathogenic variant in ceramide synthases has been identified in human patients and knockout of various ceramide synthases in mice has not led to photoreceptor degeneration. Methods Exome sequencing was used to identify candidate disease genes in patients with vision loss as confirmed by standard evaluation methods, including electroretinography (ERG) and optical coherence tomography. The vision loss phenotype in mice was evaluated by ERG and histological analyses. Results Here we have identified four patients with cone-rod dystrophy or maculopathy from three families carrying pathogenic variants in TLCD3B . Consistent with the phenotype observed in patients, the Tlcd3b KO/KO mice exhibited a significant reduction of the cone photoreceptor light responses, thinning of the outer nuclear layer, and loss of cone photoreceptors across the retina. Conclusion Our results provide the first link between loss-of-function variants in a ceramide synthase gene and human retinal dystrophy. Establishment of the Tlcd3b knockout murine model, the first in vivo photoreceptor cell degeneration model due to loss of a ceramide synthase, will provide a unique opportunity in probing the role of ceramide in survival and function of photoreceptor cells.
Previous in vitro studies indicate that CWC27 functions as a splicing factor in the Bact spliceosome complex, interacting with CWC22 to form a landing platform for eIF4A3, a core component of the exon junction complex. However, the function of CWC27 as a splicing factor has not been validated in any in vivo systems. CWC27 variants have been shown to cause autosomal recessive retinal degeneration, in both syndromic and non-syndromic forms. The Cwc27K338fs/K338fs mouse model was shown to have significant retinal dysfunction and degeneration by 6 months of age. In this report, we have taken advantage of the Cwc27K338fs/K338fs mouse model to show that Cwc27 is involved in splicing in vivo in the context of the retina. Bulk RNA and single cell RNA-sequencing of the mouse retina showed that there were gene expression and splicing pattern changes, including alternative splice site usage and intron retention. Positive staining for CHOP suggests that ER stress may be activated in response to the splicing pattern changes and is a likely contributor to the disease mechanism. Our results provide the first evidence that CWC27 functions as a splicing factor in an in vivo context. The splicing defects and gene expression changes observed in the Cwc27K338fs/K338fs mouse retina provide insight to the potential disease mechanisms, paving the way for targeted therapeutic development.
Much of the complexity of the eukaryotic cell transcriptome is due to the alternative splicing of mRNA. However, knowledge on how transcriptome complexity is translated into functional complexity remains limited. For example, although different isoforms of a gene may show distinct temporal and spatial expression patterns, it is largely unknown whether these isoforms encode proteins with distinct functions matching their expression pattern. In this report, we investigated the function and relationship of the two isoforms of Reep6, namely Reep6.1 and Reep6.2, in rod photoreceptor cells. These two isoforms result from the alternative splicing of exon 5 and show mutually exclusive expression patterns. Reep6.2 is the canonical isoform that is expressed in non-retinal tissues while Reep6.1 is the only expressed isoform in the adult retina. The Reep6.1 isoform-specific knockout mouse, Reep6E5/E5, is generated by deleting exon 5 and a homozygous deletion phenotypically displayed a rod degeneration phenotype comparable to a Reep6 full knockout mouse, indicating that the Reep6.1 isoform is essential for the rod photoreceptor cell survival. Consistent with the results obtained from a loss-of-function experiment, overexpression of Reep6.2 failed to rescue the rod degeneration phenotype of Reep6 knockout mice while overexpression of Reep6.1 does lead to rescue. These results demonstrate that, consistent with the expression pattern of the isoform, Reep6.1 has rod-specific functions that cannot be substituted by its canonical isoform. Our findings suggested that a strict regulation of splicing is required for the maintenance of photoreceptor cells.
Hereditary retinal dystrophy is clinically defined as a broad group of chronic and progressive disorders that affect visual function by causing photoreceptor degeneration. Previously, we identified mutations in the gene encoding receptor expression-enhancing protein 6 (REEP6), in individuals with autosomal recessive retinitis pigmentosa (RP), the most common form of inherited retinal dystrophy. One individual was molecularly diagnosed with biallelic REEP6 mutations, a missense mutation over a frameshift mutation. In this study, we generated Reep6 compound heterozygous mice, Reep6, which mimic the patient genotype and recapitulate the early-onset retinal degeneration phenotypes observed in the individual with RP. To determine the feasibility of rescuing the Reep6 mutant phenotype via gene replacement therapy, we delivered Reep6.1, the mouse retina-specific isoform of REEP6, to photoreceptors of Reep6 mutant mice on postnatal day 20. Evaluation of the therapeutic effects 2 months posttreatment showed improvements in the photoresponse as well as preservation of photoreceptor cells. Importantly, guanylyl cyclase 1 (GC1) expression was also restored to the outer segment after treatment. Furthermore, rAAV8-Reep6.1 single treatment in Reep6 mutant mice 1 year postinjection showed significant improvements in retinal function and morphology, suggesting that the treatment is effective even after a prolonged period. Findings from this study show that gene replacement therapy in the retina with rAAV overexpressing Reep6 is effective, preserving photoreceptor function in Reep6 mutant mice. These findings provide evidence that rAAV8-based gene therapy can prolong survival of photoreceptors in vivo and can be potentially used as a therapeutic modality for treatment of patients with RP.
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