Tcf4 CTG Repeat Expansion Induces RNA Toxicity and MBNL-Associated Splicing Dysregulation in a Mouse Model of Fuchs Endothelial Corneal Dystrophy.

PURPOSE: To determine whether a Tcf4 CTG repeat knock-in mouse model of Fuchs endothelial corneal dystrophy (FECD) recapitulates molecular features of repeat RNA toxicity and MBNL-associated splicing dysregulation in corneal endothelial cells in vivo.

METHODS: Corneal endothelial tissues from 20-, 40-, and 60-week-old wild-type and Tcf4(CTG)100/(CTG)100 mice were examined by contact specular microscopy and CUG-repeat RNA FISH with MBNL1 immunostaining. At 60 weeks, we used RT-PCR, quantitative PCR, Western blotting, and RNA-Seq analysis to assess Mbnl autoregulatory splicing, genome-wide alternative splicing, RNA-binding protein motif enrichment, and Tcf4 isoform composition; selected splicing events were compared with human FECD data.

RESULTS: Tcf4(CTG)100/(CTG)100 mice showed neither obvious guttae-like abnormalities nor nuclear RNA foci at 20 weeks; both were detected at 40 and 60 weeks, whereas wild-type mice showed neither at any age. Foci-positive nuclei and RNA foci-MBNL1 colocalization did not differ between 40 and 60 weeks. Although Mbnl1 and Mbnl2 transcript levels and MBNL1 and MBNL2 protein levels were unchanged, Mbnl2 exon 6 inclusion was reduced. RNA-Seq reanalysis identified 645 differential alternative splicing events, predominantly skipped exons, with MBNL1 motif enrichment among events with increased exon skipping. Representative MBNL-regulated events were only partially conserved between the mouse model and human FECD. Total Tcf4 expression was unchanged, whereas Tcf4-225 was selectively upregulated.

CONCLUSIONS: This model provides in vivo evidence that Tcf4 CTG repeat expansion can induce molecular hallmarks of repeat RNA toxicity and downstream MBNL-associated splicing dysregulation, together with selective Tcf4 isoform alteration. It offers an in vivo platform for mechanistic and therapeutic studies.