This is a multiple PI R01 application to test the hypothesis that trinucleotide repeat expansion (TNR) is a causal event in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD). FECD is a common, degenerative disease of the corneal endothelial cell monolayer. Unfortunately, there are no medical therapies to halt disease progression, and the pathogenesis is not well understood. As a result, surgical transplantation of the cornea is the only viable treatment. In fact, end-stage FECD is the most common cause of allogeneic corneal transplantation in the U.S., responsible for greater than 14,000 grafts annually. Recently, we have identified a CTG TNR expansion sequence within the third intron of the transcription factor 4 (TCF4) gene that associates with 79% of FECD cases. To date, this is the most predictive genetic anomaly identified for FECD. In FECD, CTG TNR expansions range from 50-2000 repeats, which are significantly higher than non-FECD patients who typically have 12-18 CTG repeats. How this TCF4 CTG TNR expansion region promotes the FECD phenotype is not clear. Similar expansion of unstable TNR sequences have been identified as the causative pathogenic mechanism in several rare and debilitating neurologic disorders, such as spin cerebellar ataxias, Huntington disease, fragile X syndrome and myotonic dystrophy, type 1. These TNR expansion regions are thought to interfere with normal protein function, expression of the TNR containing gene, and/or RNA splicing in cis and trans. In addition, translation of toxic Repeat-Associated Non-ATG (RAN) translation products from the sense and antisense transcripts of the TNR expansion sequences have also been identified as a contributing factor towards disease progression. Our preliminary studies suggest similar events are occurring in FECD cells. According to our data, transcripts from the TCF4 CTG TNR expansion region are retained in the nucleus and form ribonuclear inclusions called RNA foci in FECD tissue and primary FECD monolayer cells. These RNA foci sequester the splicing factor MBNL1, leading to altered splicing of numerous transcripts. We also have evidence for RAN translation of TCF4 CTG TNR expansion RNA, producing small homopolymeric protein aggregates in corneal endothelial cells of FECD patients. Our central hypothesis is that RNA foci and RAN translation protein products arising from the TCF4 CTG TNR expansion region causes FECD by altering gene expression and disrupting critical cell functions. It is our premise that stress factors (e.g. oxidative stress) promote instability in the TCF4 CTG TNR expansion sequence resulting in the formation of CUG repeat containing RNA foci and accumulation of RAN translation products. The presence of these products leads to differential mRNA splicing and altered expression of critical genes, stimulating ER stress-mediated activation of unfolded protein response (UPR), endothelial to mesenchymal transformation (EMT) and disordered extracellular matrix (ECM) deposition. We will test our hypothesis with two specific aims - 1) Characterize the molecular anomalies associated with TCF4 CTG TNR expansion in FECD; 2) Determine the role of TCF4 CTG TNR expansion in pathogenesis of FECD. Completion of these two specific aims will provide a mechanistic understanding of how TCF4 CTG TNR expansion is involved in FECD pathogenesis. Understanding the molecular events underlying the pathogenesis of FECD will be essential for future development of targeted therapeutic strategies for management of FECD.
The TCF4 CTG TNR expansion sequence associates with 79% of Fuchs endothelial corneal dystrophy (FECD) patients and is by far the most predictive genomic anomaly associated with the disease. The proposed studies will yield essential fundamental information regarding the mechanisms involved with TCF4 CTG TNR expansion and the development of the FECD phenotype. Analysis of primary FECD and control corneal endothelial cell cultures with varying expansion lengths will be used to investigate development of novel ribonuclear inclusions called RNA foci and Repeat-Associated Non-ATG (RAN) translation products derived from the sense and antisense transcripts of the TCF4 CTG TNR expansion sequences. Utilization of novel cell-based model systems will enable us to investigate how these mechanisms create physiologic changes in cell functions. Completion of this proposal will provide a comprehensive understanding of FECD pathogenesis and identification of novel target molecules to which future therapies can be directed.
|Wieben, Eric D; Aleff, Ross A; Tang, Xiaojia et al. (2017) Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy. Invest Ophthalmol Vis Sci 58:343-352|
|Roy Chowdhury, Uttio; Rinkoski, Tommy A; Bahler, Cindy K et al. (2017) Effect of Cromakalim Prodrug 1 (CKLP1) on Aqueous Humor Dynamics and Feasibility of Combination Therapy With Existing Ocular Hypotensive Agents. Invest Ophthalmol Vis Sci 58:5731-5742|