Shivakumar Vasanth

Mutations in AGBL1 Cause Dominant Late-Onset Fuchs Corneal Dystrophy and Alter Protein-Protein Interaction with TCF4

Fuchs corneal dystrophy (FCD) is a hereditary dystrophy of the corneal endothelium and is responsible for majority of the corneal transplantation performed in the United States. Here, we describe three generations of a family with 12 individuals affected by late-onset FCD and in which three individuals are unaffected. Genome-wide mapping provided suggestive linkage at two loci on chromosomal arms 3p and 15q. Alleles at either locus alone were not sufficient to explain FCD; however, considered together, both loci could explain the disorder in this pedigree. Subsequent next-generation sequencing identified a nonsense mutation in AGBL1 in the 15q locus; this mutation would result in a premature termination of AGBL1. Consistent with a causal role for this transcript, further sequencing of our cohort of late-onset-FCD-affected individuals identified two cases harboring the same nonsense mutation and a further three unrelated individuals bearing a second missense allele. AGBL1 encodes a glutamate decarboxylase previously identified in serial analysis of gene expression of corneal endothelium, a finding confirmed by immunohistochemical staining. Wild-type AGBL1 localizes predominantly to the cytoplasm; in sharp contrast, the truncated protein showed distinct nuclear localization. Finally, we show that AGBL1 interacts biochemically with the FCD-associated protein TCF4 and that the mutations found in our cohort of FCD individuals diminish this interaction. Taken together, our data identify a locus for FCD, extend the complex genetic architecture of the disorder, provide direct evidence for the involvement of TCF4 in FCD pathogenesis, and begin to explain how causal FCD mutations affect discrete biochemical complexes.

Metabolic Regulation and Energy Homeostasis through the Primary Cilium

Obesity and diabetes represent a significant healthcare concern. In contrast to genome-wide association studies that, some exceptions notwithstanding, have offered modest clues about pathomechanism, the dissection of rare disorders in which obesity represents a core feature have highlighted key molecules and structures critical to energy regulation. Here we focus on the primary cilium, an organelle whose roles in energy homeostasis have been underscored by the high incidence of obesity and type II diabetes in patients and mouse mutants with compromised ciliary function. We discuss recent evidence linking ciliary dysfunction to metabolic defects and we explore the contribution of neuronal and nonneuronal cilia to these phenotypes.

Activation of mitophagy leads to decline in Mfn2 and loss of mitochondrial mass in Fuchs endothelial corneal dystrophy

Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.

CTG18.1 Expansion in TCF4 Increases Likelihood of Transplantation in Fuchs Corneal Dystrophy.

Purpose: Fuchs dystrophy is the leading indication of corneal transplantation in the United States. A CTG18.1 trinucleotide repeat in TCF4 correlates with increased severity in Fuchs dystrophy; however, quantitative estimates of increased transplantation risk, including effects of age and sex, are unclear. Methods: In a tertiary institution clinical practice, 574 participants were enrolled in a longitudinal study of Fuchs dystrophy after slit-lamp biomicroscopy confirmed significant central guttae and/or corneal transplantation in both eyes. We documented clinical history, examination findings, and demographic information. We acquired blood samples, extracted DNA, and sequenced the CTG18.1 trinucleotide repeat in TCF4. In this retrospective case–control study, the number of participants with triplet expansion, defined as greater than 40 CTG repeats, and transplantation status were assessed. Kaplan–Meier estimates of timing and transplantation events were produced. The Cox proportional hazard regression model was used to assess the relationship between age, sex, triplet expansion, and surgery. Results: A total of 106 participants (18.5%) previously underwent corneal transplantation in at least 1 eye at the time of initial evaluation. A higher proportion of individuals harboring allele expansion had undergone transplantation (78/357, 21.8%) compared with those without the expanded allele (28/217, 12.9%), a significant association (P = 0.007). The log-rank test demonstrates a significant difference in survival function over time (P = 0.027), with a hazard ratio of 1.64 (95% confidence interval, 1.05–2.55). Conclusions: Expansion of the TCF4 CTG trinucleotide repeat was associated with 1.64 times higher likelihood of corneal transplantation at a given age in patients with Fuchs dystrophy.

NQO1 downregulation potentiates menadione-induced endothelial-mesenchymal transition during rosette formation in Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a genetic and oxidative stress disorder of post-mitotic human corneal endothelial cells (HCEnCs), which normally exhibit hexagonal shape and form a compact monolayer compatible with normal corneal functioning and clear vision. FECD is associated with increased DNA damage, which in turn leads to HCEnC loss, resulting in the formation rosettes and aberrant extracellular matrix (ECM) deposition in the form of pro-fibrotic guttae. Since the mechanism of ECM deposition in FECD is currently unknown, we aimed to investigate the role of endothelial-mesenchymal transition (EMT) in FECD using a previously established cellular in vitro model that recapitulates the characteristic rosette formation, by employing menadione (MN)-induced oxidative stress. We demonstrate that MN treatment alone, or a combination of MN and TGF-β1 induces reactive oxygen species (ROS), cell death, and EMT in HCEnCs during rosette formation, resulting in upregulation of EMT- and FECD-associated markers such as Snail1, N-cadherin, ZEB1, and transforming growth factor-beta-induced (TGFβI), respectively. Additionally, FECD ex vivo specimens displayed a loss of organized junctional staining of plasma membrane-bound N-cadherin, with corresponding increase in fibronectin and Snail1 compared to ex vivo controls. Addition of N-acetylcysteine (NAC) downregulated all EMT markers and abolished rosette formation. Loss of NQO1, a metabolizing enzyme of MN, led to greater increase in intracellular ROS levels as well as a significant upregulation of Snail1, fibronectin, and N-cadherin compared to normal cells, indicating that NQO1 regulates Snail1-mediated EMT. This study provides first line evidence that MN-induced oxidative stress leads to EMT in corneal endothelial cells, and the effect of which is further potentiated when redox cycling activity of MN is enhanced by the absence of NQO1. Given that NAC inhibits Snail-mediated EMT, this may be a potential therapeutic intervention for FECD.

Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells

Purpose Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. Methods We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry–based proteome sequencing. Results The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry–based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. Conclusions We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.