Existence of Neural Crest-Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium.

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2ß), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, ß-III tubulin, and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies.

Mutations in LOXHD1, a recessive-deafness locus, cause dominant late-onset Fuchs corneal dystrophy.

Fuchs corneal dystrophy (FCD) is a genetic disorder of the corneal endothelium and is the most common cause of corneal transplantation in the United States. Previously, we mapped a late-onset FCD locus, FCD2, on chromosome 18q. Here, we present next-generation sequencing of all coding exons in the FCD2 critical interval in a multigenerational pedigree in which FCD segregates as an autosomal-dominant trait. We identified a missense change in LOXHD1, a gene causing progressive hearing loss in humans, as the sole variant capable of explaining the phenotype in this pedigree. We observed LOXHD1 mRNA in cultured human corneal endothelial cells, whereas antibody staining of both human and mouse corneas showed staining in the corneal epithelium and endothelium. Corneal sections of the original proband were stained for LOXHD1 and demonstrated a distinct increase in antibody punctate staining in the endothelium and Descemet membrane; punctate staining was absent from both normal corneas and FCD corneas negative for causal LOXHD1 mutations. Subsequent interrogation of a cohort of >200 sporadic affected individuals identified another 15 heterozygous missense mutations that were absent from >800 control chromosomes. Furthermore, in silico analyses predicted that these mutations reside on the surface of the protein and are likely to affect the protein's interface and protein-protein interactions. Finally, expression of the familial LOXHD1 mutant allele as well as two sporadic mutations in cells revealed prominent cytoplasmic aggregates reminiscent of the corneal phenotype. All together, our data implicate rare alleles in LOXHD1 in the pathogenesis of FCD and highlight how different mutations in the same locus can potentially produce diverse phenotypes.

Structure of the tandem MA-3 region of Pdcd4 protein and characterization of its interactions with eIF4A and eIF4G: molecular mechanisms of a tumor suppressor.

One of the key regulatory points of translation initiation is recruitment of the 43S preinitation complex to the 5' mRNA cap by the eIF4F complex (eIF4A, eIF4E, and eIF4G). The tumor suppressor protein Pdcd4 has been shown to inhibit cap-dependent translation by interacting tightly with the RNA helicase eIF4A via its tandem MA-3 domains. The NMR studies reported here reveal a fairly extensive and well defined interface between the two MA-3 domains in solution, which appears to be stabilized by a network of interdomain salt bridges and hydrogen bonds, and reveals a unique orientation of the two domains. Characterization of the stoichiometry of the Pdcd4-eIF4A complex suggests that under physiological conditions Pdcd4 binds to a single molecule of eIF4A, which involves contacts with both Pdcd4 MA-3 domains. We also show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eIF4A in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo. The formation of a 1:1 eIF4A-Pdcd4 complex in solution is consistent with the reported presence in vivo of only one molecule of eIF4A in the eIF4F complex. Pdcd4 has also been reported to interact directly with the middle region of eIF4G, however, we were unable to obtain any evidence for even a weak, transient direct interaction.

Molecular bases of corneal endothelial dystrophies.

The phrase "corneal endothelial dystrophies" embraces a group of bilateral corneal conditions that are characterized by a non-inflammatory and progressive degradation of corneal endothelium. Corneal endothelial cells exhibit a high pump site density and, along with barrier function, are responsible for maintaining the cornea in its natural state of relative dehydration. Gradual loss of endothelial cells leads to an insufficient water outflow, resulting in corneal edema and loss of vision. Since the pathologic mechanisms remain largely unknown, the only current treatment option is surgical transplantation when vision is severely impaired. In the past decade, important steps have been taken to understand how endothelial degeneration progresses on the molecular level. Studies of affected multigenerational families and sporadic cases identified genes and chromosomal loci, and revealed either Mendelian or complex disorder inheritance patterns. Mutations have been detected in genes that carry important structural, metabolic, cytoprotective, and regulatory functions in corneal endothelium. In addition to genetic predisposition, environmental factors like oxidative stress were found to be involved in the pathogenesis of endotheliopathies. This review summarizes and crosslinks the recent progress on deciphering the molecular bases of corneal endothelial dystrophies.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

AIMS: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. RESULTS: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. INNOVATION: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. CONCLUSION: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

Fuchs endothelial cornea dystrophy: a review of the genetics behind disease development.

Fuchs dystrophy represents the most common form of endothelial dystrophy and is a significant cause of visual impairment. The cause of Fuchs dystrophy is a complicated combination of both genetic and environmental factors. Understanding the underlying causes of the disease can potentially lead to new medical treatments preventing loss of vision.

Sulforaphane decreases endothelial cell apoptosis in fuchs endothelial corneal dystrophy: a novel treatment.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is an oxidative stress disorder that leads to age-related and gradual loss of corneal endothelial cells resulting in corneal edema and loss of vision. To date, other than surgical intervention, there are no treatment options for patients with FECD. We have shown that in FECD, there is a deficiency in nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated antioxidant defense due to decreased Nrf2 nuclear translocation and activation of antioxidant response element (ARE). In this study, we used sulforaphane (SFN) and D3T to investigate a strategy of targeting Nrf2-ARE in FECD. METHODS: FECD and normal ex vivo corneas and human corneal endothelial cell lines were pretreated with SFN or D3T and exposed to oxidative stress with tert-Butyl hydroperoxide (tBHP). Apoptosis was detected with TUNEL. Cellular localization of Nrf2 and p53 was assessed by immunohistochemistry. Effect of SFN was determined by using DCFDA assay, Western blot and real-time PCR. RESULTS: After pretreatment with SFN, oxidative stress was induced with tBHP. In ex vivo FECD specimens, SFN decreased CEC apoptosis by 55% in unstressed group and by 43% in tBHP-treated specimens. SFN enhanced nuclear translocation of Nrf2 in FECD specimens and decreased p53 staining under oxidative stress. Pretreatment with SFN enhanced cell viability by decreasing intracellular reactive oxygen species production. Upregulation of Nrf2 levels led to increased synthesis of DJ-1, heme oxygenase 1, and nicotinamide adenine dinucleotide quinone oxidoreductase-1. SFN significantly upregulated major ARE-dependent antioxidants and ameliorated oxidative stress-induced apoptosis in FECD. CONCLUSIONS: Our results suggest that targeting Nrf2-ARE pathway may arrest degenerative cell loss seen in FECD.

Decline in DJ-1 and decreased nuclear translocation of Nrf2 in Fuchs endothelial corneal dystrophy.

PURPOSE: This study sought to determine factors involved in nuclear factor erythroid 2-related factor 2 (Nrf2) regulation and their response to oxidative stress in Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs). METHODS: FECD corneal buttons were obtained from transplantations and normal human corneas from tissue banks. Oxidative stress was induced by tert-butyl hydroperoxide (tBHP). Protein and mRNA levels of Nrf2, DJ-1, p53, and Kelch-like ECH-associated protein1 (Keap1) were investigated using Western blotting and real-time PCR. Immunoprecipitation was used to detect levels of oxidized DJ-1 protein and Cullin 3- (Cul3)-regulated degradation of DJ-1 in immortalized FECD (FECDi) and normal CEC (HCECi) cell lines. Nrf2 subcellular localization was assessed by immunocytochemistry. RESULTS: Nrf2 protein stabilizer, DJ-1, decreased significantly in FECD CECs compared with normal, whereas Nrf2 protein repressor, Keap1, was unchanged at baseline but increased under oxidative stress. Under oxidative stress, normal CECs upregulated DJ-1 protein synthesis, whereas FECD CECs did not. DJ-1 decline correlated with increased DJ-1 oxidative modification and carbonylation in FECDi as compared with HCECi. Increased labeling of immunoprecipitated DJ-1 protein with anti-Cul3 antibody indicated enhanced DJ-1 degradation in FECDi as compared with HCECi. Following tBHP treatment, Nrf2 translocated from cytoplasm to nuclei in normal CECs, whereas Nrf2 nuclear localization was not observed in FECD. CONCLUSIONS: Decreased levels of DJ-1 in FECD at baseline and under oxidative stress correlate with impaired Nrf2 nuclear translocation and heightened cell susceptibility to apoptosis. Targeting the DJ-1/Nrf2 axis could yield a mechanism to slow CEC degeneration in FECD.

Telomerase immortalization of human corneal endothelial cells yields functional hexagonal monolayers.

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness-the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.

B-Myb promotes S-phase independently of its sequence-specific DNA binding activity and interacts with polymerase delta-interacting protein 1 (Pdip1).

B-Myb is a highly conserved member of the Myb transcription factor family, which plays an essential role in cell cycle progression by regulating the transcription of genes at the G 2/M-phase boundary. The role of B-Myb in other parts of the cell cycle is less well-understood. By employing siRNA-mediated silencing of B-Myb expression, we found that B-Myb is required for efficient entry into S-phase. Surprisingly, a B-Myb mutant that lacks sequence-specific DNA-binding activity and is unable to activate transcription of B-Myb target genes is able to rescue the S-phase defect observed after B-Myb knockdown. Moreover, we have identified polymerase delta-interacting protein 1 (Pdip1), a BTB domain protein known to bind to the DNA replication and repair factor PCNA as a novel B-Myb interaction partner. We have shown that Pdip1 is able to interact with B-Myb and PCNA simultaneously. In addition, we found that a fraction of endogenous B-Myb can be co-precipitated via PCNA, suggesting that B-Myb might be involved in processes related to DNA replication or repair. Taken together, our work suggests a novel role for B-Myb in S-phase that appears to be independent of its sequence-specific DNA-binding activity and its ability to stimulate the expression of bona fide B-Myb target genes.

p53-regulated increase in oxidative-stress--induced apoptosis in Fuchs endothelial corneal dystrophy: a native tissue model.

PURPOSE: This study compared susceptibility of Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs) to oxidative stress, and studied the mechanism of oxidative-stress-induced apoptosis in FECD-affected endothelium. METHODS: For in vitro studies, immortalized normal and FECD human corneal endothelial cell lines (HCECi and FECDi, respectively) were exposed to tert-butyl hydroperoxide (tBHP). Apoptotic cell populations were distinguished using flow cytometry. Reactive oxygen species production was measured by a horseradish peroxidase assay. For ex vivo studies, CECs were exposed to tBHP. Oxidative DNA damage and apoptosis were assessed by anti-8-hydroxydeoxyguanosine antibody and TUNEL assay, respectively. p53 and phospho-p53 levels were assessed by Western blot and immunohistochemistry. RESULTS: Flow cytometry revealed a higher rate of apoptosis in FECDi than that in HCECi after exposure to 0.5 mM (P=0.010) and 1.0 mM tBHP (P=0.041). Further analysis showed increased production of H2O2 by FECDi than that by HCECi. Oxidative DNA damage increased in both normal and FECD CECs after exposure to 0.5 mM tBHP (P=0.031 and 0.022, respectively), leading to a 21% increase in TUNEL-positive CECs in FECD (P=0.015) but no change in normal. Baseline p53 expression was twofold higher in FECD than that in normal endothelium (P=0.002). Immunofluorescence revealed an increase in p53 and phospho-p53 levels in FECD compared with that in normal endothelium. CONCLUSIONS: FECD CECs are more susceptible to oxidative DNA damage and oxidative-stress-induced apoptosis than normal. Increased activation of p53 in FECD suggests that it mediates cell death in susceptible CECs. The authors conclude that p53 plays a critical role in complex mechanisms regulating oxidative-stress-induced apoptosis in FECD.

Resonance assignment and secondary structure of the middle MA-3 domain and complete tandem MA-3 region of the tumour suppressor protein Pdcd4.

Pdcd4 (Programmed Cell Death Protein 4) is a novel eukaryotic tumour suppressor protein, which is involved in the regulation of both transcription and translation (reviewed in Lankat-Buttgereit and Göke 2009). The protein contains two interacting MA-3 domains (MA-3(M) and MA-3(C)), which are linked by a short semi-flexible linker region (Waters et al. 2007; Suzuki et al. 2008). The MA-3 domains are involved in mediating specific protein-protein interactions with functional partners such as eIF4A (Yang et al. 2003 ). Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a construct composed of the middle MA-3 domain and subsequent linker region (MA-3(M)) and backbone assignments for the entire tandem MA-3 region of Pdcd4 (Pdcd4 MA-3(M-C)). Analysis of the backbone chemical shift data obtained indicates that Pdcd4 MA-3(M) contains eight helical regions corresponding to over 74% of the protein backbone and that Pdcd4 MA-3(M-C) contains fifteen helical regions (72%). Comparison of the position of these helical regions with those observed in the crystal structures suggests that the solution and crystal structures of both proteins are very similar.