Ophthalmic drug effects on the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment.

Drugs that can treat one disease may either be detrimental or beneficial toward another due to possible cross-interactions. Therefore, care in choosing a suitable drug for patients with multiple diseases is crucial in successful patient management. This study explores several currently available ophthalmic drugs used to treat common ocular diseases to understand how they can affect the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment found in abundance in the corneal protein aggregation deposits of lattice corneal dystrophy (LCD) patients. Results from this study provided supporting evidence that some drugs intended to treat other diseases can enhance or inhibit fibrillar aggregation of TGFBIp peptide, which may have potential implication of affecting the disease progression of LCD by either worsening or ameliorating it. Comparisons of the different properties of ophthalmic compounds explored in this study may also provide some guidance for future design of drugs geared toward the treatment of LCD.

Uncovering the role of ferroptosis in Bietti crystalline dystrophy and potential therapeutic strategies.

PURPOSE: Bietti crystalline dystrophy (BCD) is an inherited retinal degeneration disease caused by mutations in the CYP4V2 gene. Currently, there is no clinical therapy approach available for BCD patients. Previous research has suggested that polyunsaturated fatty acids (PUFAs) may play a significant role in the development of BCD, implicating the involvement of ferroptosis in disease pathogenesis. In this work, we aimed to investigate the interplay between ferroptosis and BCD and to detect potential therapeutic strategies for the disease. METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype. RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice. CONCLUSION: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

Defective cell adhesion function of solute transporter, SLC4A11, in endothelial corneal dystrophies.

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.

Schnyder corneal dystrophy-associated UBIAD1 mutations cause corneal cholesterol accumulation by stabilizing HMG-CoA reductase.

Schnyder corneal dystrophy (SCD) is a rare genetic eye disease characterized by corneal opacification resulted from deposition of excess free cholesterol. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is an enzyme catalyzing biosynthesis of coenzyme Q10 and vitamin K2. More than 20 UBIAD1 mutations have been found to associate with human SCD. How these mutants contribute to SCD development is not fully understood. Here, we identified HMGCR as a binding partner of UBIAD1 using mass spectrometry. In contrast to the Golgi localization of wild-type UBIAD1, SCD-associated mutants mainly resided in the endoplasmic reticulum (ER) and competed with Insig-1 for HMGCR binding, thereby preventing HMGCR from degradation and increasing cholesterol biosynthesis. The heterozygous Ubiad1 G184R knock-in (Ubiad1G184R/+) mice expressed elevated levels of HMGCR protein in various tissues. The aged Ubiad1G184R/+ mice exhibited corneal opacification and free cholesterol accumulation, phenocopying clinical manifestations of SCD patients. In summary, these results demonstrate that SCD-associated mutations of UBIAD1 impair its ER-to-Golgi transportation and enhance its interaction with HMGCR. The stabilization of HMGCR by UBIAD1 increases cholesterol biosynthesis and eventually causes cholesterol accumulation in the cornea.

0.1% RGN-259 (Thymosin ß4) Ophthalmic Solution Promotes Healing and Improves Comfort in Neurotrophic Keratopathy Patients in a Randomized, Placebo-Controlled, Double-Masked Phase III Clinical Trial.

We determined the efficacy and safety of 0.1% RGN-259 ophthalmic solution (containing the regenerative protein thymosin ß4) in promoting the healing of persistent epithelial defects in patients with Stages 2 and 3 neurotrophic keratopathy. Complete healing occurred after 4 weeks in 6 of the 10 RGN-259-treated subjects and in 1 of the 8 placebo-treated subjects (p = 0.0656), indicating a strong efficacy trend. Additional efficacy was seen in the significant healing (p = 0.0359) with no recurrent defects observed at day 43, two weeks after cessation of treatment, while the one healed placebo-treated subject at day 28 suffered a recurrence at day 43. The Mackie classification disease stage improved in the RGN-259-treated group at Days 29, 36, and 43 (p = 0.0818, 0.0625, and 0.0467, respectively). Time to complete healing also showed a trend towards efficacy (p = 0.0829, Kaplan-Meier) with 0.1% RGN-259. RGN-259-treated subjects had significant improvements at multiple time points in ocular discomfort, foreign body sensation, and dryness which were not seen in the placebo group. No significant adverse effects were observed. In summary, the use of 0.1% RGN-259 promotes rapid healing of epithelial defects in neurotrophic keratopathy, improves ocular comfort, and is safe for treating this challenging population of patients.

Different mRNA expression patterns in keratoglobus and pellucid marginal degeneration keratocytes.

PURPOSE: Alike keratoconus (KC), keratoglobus (KG) and pellucid marginal degeneration (PMD) belong to ectatic corneal diseases. While there are numerous studies on keratoconus pathophysiology, there is no exact knowledge on genetic and pathophysiological background of KG and PMD, so far. It is not yet clarified, whether KG and PMD are independent clinical entities or represent different stages of the same disease. Our purpose was to investigate key parameters concerning collagen synthesis, intracellular LOX expression and inflammation in corneal stromal cells of KG and PMD subjects, in vitro. METHODS: Normal human keratocytes of corneas from the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz and human keratocytes of KG and PMD patients were isolated and cultured as keratocytes. To examine Collagen I and V (Col I, Col V), heat shock protein 47 (Hsp47), Lysyl Oxidase (LOX), nuclear factor kappa B (NF-κB) mRNA and protein expression in all cell types, quantitative PCR and Western blot analysis has been performed. RESULTS: Col5A1 mRNA expression was significantly lower in KG and PMD keratocytes and LOX mRNA expression was significantly higher in KG-keratocytes, compared to controls. Col1A1, Hsp47 and NF-κB mRNA expression and the analyzed protein expressions did not differ from controls, in KG or PMD. CONCLUSIONS: Col5A1 mRNA expression is decreased in KG and PMD and LOX mRNA expression is increased in KG. Therefore, the pathophysiology of KG and PMD differs from KC and these seem to be from KC independent entities. The explanation of the peripheral corneal thinning in KG and PMD must be investigated in further studies.

PSCs Reveal PUFA-Provoked Mitochondrial Stress as a Central Node Potentiating RPE Degeneration in Bietti's Crystalline Dystrophy.

Bietti's crystalline dystrophy (BCD) is an incurable retinal disorder caused by the polypeptide 2 of cytochrome P450 family 4 subfamily V (CYP4V2) mutations. Patients with BCD present degeneration of retinal pigmented epithelial (RPE) cells and consequent blindness. The lack of appropriate disease models and patients' RPE cells limits our understanding of the pathological mechanism of RPE degeneration. In this study, using CYP4V2 mutant pluripotent stem cells as disease models, we demonstrated that RPE cells with CYP4V2 mutations presented a disrupted fatty acid homeostasis, which were characterized with excessive accumulation of poly-unsaturated fatty acid (PUFA), including arachidonic acid (AA) and eicosapentaenoic acid (EPA). The PUFA overload increased mitochondrial reactive oxygen species, impaired mitochondrial respiratory functions, and triggered mitochondrial stress-activated p53-independent apoptosis in CYP4V2 mutant RPE cells. Restoration of the mutant CYP4V2 using adeno-associated virus 2 (AAV2) can effectively reduce PUFA deposition, alleviate mitochondria oxidative stresses, and rescue RPE cell death in BCD RPE cells. Taken together, our results highlight a role of PUFA-induced mitochondrial damage as a central node to potentiate RPE degeneration in BCD patients. AAV2-mediated gene therapy may represent a feasible strategy for the treatment of BCD.

Reduction of lipid accumulation rescues Bietti's crystalline dystrophy phenotypes.

Bietti's crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

Genotypic Homogeneity in Distinctive Transforming Growth Factor-Beta Induced (TGFBI) Protein Phenotypes.

Background: To evaluate the distribution of the transforming growth factor-beta induced (TGFBI) corneal dystrophies in a multi-ethnic population in Singapore, and to present the different phenotypes with the same genotype. Methods: This study included 32 patients. Slit lamp biomicroscopy was performed for each patient to determine the disease phenotype. Genomic DNA was extracted from the blood samples and the 17 exons of the TGFBI gene were amplified by PCR and sequenced bi-directionally for genotype analysis. Results: Regarding phenotypes, the study patients comprised 11 (34.4%; 8 with R555W and 3 with R124H mutation) patients with granular corneal dystrophy type 1 (GCD1), 6 (18.8%; 5 with R124H and 1 with R124C mutation) patients with GCD2, 13 (40.6%; 7 with R124C, 2 with H626R, 2 with L550P, 1 with A620D and 1 with H572R) patients with lattice corneal dystrophy (LCD) and 2 (6.3%; 1 with R124L and 1 with R124C) patients with Reis-Bückler corneal dystrophy. Regarding genotype, R124H mutation was associated with GCD2 (5 cases; 62.5%) and GCD1 (3 cases; 37.5%). R124C mutation was associated with LCD (7 cases; 87.5%) and GCD2 (1 case; 12.5%). All the 8 cases (100%) of R555W mutation were associated with GCD1. Conclusions: Although the association between genotype and phenotype was good in most cases (65.7%; 21 of 32 patients), genotype/phenotype discrepancy was observed in a significant number.

pH dependence of the Slc4a11-mediated H+ conductance is influenced by intracellular lysine residues and modified by disease-linked mutations.

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.

A novel duplication of PRMD13 causes North Carolina macular dystrophy: overexpression of PRDM13 orthologue in drosophila eye reproduces the human phenotype.

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the first time in an animal model that overexpression of PRDM13 alone causes a severe abnormal retinal development. It is noteworthy that mutations associated with this autosomal dominant foveal developmental disorder are frequently duplications always including an entire copy of PRDM13, or variants in one DNase 1 hypersensitivity site at this locus.

R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells.

SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH4Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH4Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH4Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties.

The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis.

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.

Alterations in GRHL2-OVOL2-ZEB1 axis and aberrant activation of Wnt signaling lead to altered gene transcription in posterior polymorphous corneal dystrophy.

Mutations associated with posterior polymorphous corneal dystrophy (PPCD) have been identified in three genes: ZEB1 (zinc-finger E-box binding homeobox 1) associated with sub-type PPCD3; OVOL2 (ovol-like zinc finger 2) associated with sub-type PPCD1; and GRHL2 (grainyhead like transcription factor 2) associated with sub-type PPCD4. Each of these genes encodes a transcription factor that regulates cell-state transitions. While the discovery of these PPCD-associated genes has greatly expanded our knowledge of the genetic basis of PPCD, the molecular mechanisms via which mutations in these genes lead to indistinguishable disease phenotypes have yet to be elucidated. To characterize the gene expression profiles of the genetic sub-types of PPCD, RNA-seq was performed on corneal endothelium derived from an individual with PPCD1 who harbors a c.-307T > C OVOL2 promoter mutation. Transcriptomic analysis of this and previously-reported RNA-seq data from two individuals with PPCD (the first with PPCD3 associated with a ZEB1 truncating mutation (c.1381delinsGACGAT) and the second with genetically unresolved PPCD in which ZEB1 coding region, OVOL2 promoter and GRHL2 promoter, exon 1, and intron 1 mutations were excluded) revealed: OVOL2 expression increased in PPCD1 (259 fold), unchanged in PPCD3 and slightly increased in genetically unresolved PPCD (from 0 TPM to 0.86 TPM, undefined fold change); ZEB1 expression decreased in PPCD1 (-5.9 fold), PPCD3 (-3.95 fold) and genetically unresolved PPCD (-3.96 fold); and GRHL2 expression increased in PPCD1 (333.5 fold), slightly increased (from 0 TPM to 0.67 TPM, undefined fold change) in PPCD3 and increased in genetically unresolved PPCD (1853 fold). Additionally, as the majority of pedigrees affected with PPCD remain genetically unresolved, we screened the promoter, exon 1, and intron 1 regions of GRHL2 in 24 PPCD probands who do not harbor a ZEB1 or OVOL2 mutation. GRHL2 screening did not identify any novel or rare GRHL2 variant in these 24 individuals. As ZEB1 can act as an activator or repressor of downstream target gene expression depending on Wnt signaling pathway activation or deactivation, we also sought to determine whether or not Wnt signaling is active in PPCD by performing immunohistochemistry in corneal tissue sections derived from an individual affected with PPCD3 and from an individual with genetically unresolved PPCD. Immunohistochemistry results demonstrated corneal endothelial nuclear accumulation of S552 phos-ß-catenin and cytosolic localization of S33/37/T42 non-phosphorylated ß-catenin in PPCD, indicating aberrant activation of Wnt signaling, which was not observed in control corneal endothelium. These findings suggest that alterations in the ZEB1-OVOL2-GRHL2 axis (caused by PPCD-associated mutations) lead to changes in corneal endothelial cell state and molecular pathways, including the aberrant activation of the Wnt signaling pathway.

APP processing and metabolism in corneal fibroblasts and epithelium as a potential biomarker for Alzheimer's disease.

Alzheimer's disease (AD) primarily affects the brain and is the most common form of dementia worldwide. Despite more than a century of research, there are still no early biomarkers for AD. It has been reported that AD affects the eye, which is more accessible for imaging than the brain; however, links with the cornea have not been evaluated. To investigate whether the cornea could be used to identify possible diagnostic indicators of AD, we analyzed the proteolytic processing and isoforms of amyloid precursor protein (APP) and evaluated the expression of AD-related genes and proteins in corneal fibroblasts from wild-type (WT) corneas and corneas from patients with granular corneal dystrophy type 2 (GCD2), which is related to amyloid formation in the cornea. Reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the expression of AD-related genes, i.e., APP, ADAM10, BACE1, BACE2, PSEN1, NCSTN, IDE, and NEP. RT-PCR and DNA sequencing analysis demonstrated that isoforms of APP770 and APP751, but not APP695, were expressed in corneal fibroblasts. Moreover, the mRNA ratio of APP770/APP751 isoforms was approximately 4:1. Western blot analysis also demonstrated the expression of a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), beta-site APP-cleaving enzyme 1 (BACE1), nicastrin, insulin degradation enzyme, and neprilysin in corneal fibroblasts. Among these targets, the levels of immature ADAM10 and BACE1 protein were significantly increased in GCD2 cells. The expression levels of APP, ADAM10, BACE1, and transforming growth factor-beta-induced protein (TGFBIp) were also detected by western blot in human corneal epithelium. We also investigated the effects of inhibition of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems (UPS) on APP processing and metabolism. These pathway inhibitors accumulated APP, α-carboxy-terminal fragments (CTFs), ß-CTFs, and the C-terminal APP intracellular domain (AICD) in corneal fibroblasts. Analysis of microRNAs (miRNAs) revealed that miR-9 and miR-181a negatively coregulated BACE1 and TGFBIp, which was directly associated with the pathogenesis of AD and GCD2, respectively. Immunohistochemical analysis indicated that APP and BACE1 were distributed in corneal stroma cells, epithelial cells, and the retinal layer in mice. Collectively, we propose that the cornea, which is the transparent outermost layer of the eye and thus offers easy accessibility, could be used as a potential biomarker for AD diagnosis and progression.

Lithium chloride (LiCl) induced autophagy and downregulated expression of transforming growth factor ß-induced protein (TGFBI) in granular corneal dystrophy.

This study evaluated whether lithium chloride (LiCl) prevents cytoplasmic accumulation of mutant-transforming growth factor ß-induced protein (Mut-TGFBI) in granular corneal dystrophy (GCD) via activation of the autophagy pathway. Levels of TGFBI and microtubule-associated protein 1A/1B-light chain 3 (LC3) in 3 GCD patients and healthy controls were analyzed by immunohistochemistry (IHC) staining and Western blot. Primary corneal fibroblasts were isolated and transfected with wild type or mutant type TGFBI over-expressed vectors, and then treated with LiCl and/or autophagy inhibitor 3-methyladenine (3-MA). Then, levels of TGFBI, glycogen synthase kinase-3 (GSK-3) and LC3-I/-II were detected. Cell viability and transmission electron microscopy assay were also performed. Levels of TGFBI and LC3 were significantly increased in GCD patients. Over-expression of mutant type TGFBI inhibited cell viability and induced autophagy in corneal fibroblasts. LiCl downregulated the expression of TGFBI in mutant type TGFBI over-expressed cells in a dose- and time-dependent manner. LiCl enhanced autophagy in mutant type TGFBI over-expressed cells and recovered cell viability in those cells. However, the effects of LiCl were partly attenuated when autophagy was suppressed by 3-MA. To summarize, treatment with LiCl inhibited the expression of TGFBI and recovery the inhibitory of mutant type TGFBI in cell viability, at least part through enhancing of autophagy. These data strongly suggest that LiCl may be useful in the treatment of GCD.

Heredity and in vivo confocal microscopy of punctiform and polychromatic pre-Descemet dystrophy.

PURPOSE: To describe and analyze the biomicroscopic features and in vivo confocal microscopy of the crystalline form of pre-Descemet corneal dystrophy (PDCD). METHODS: We examined two non-related families using biomicroscopy, in vivo confocal microscopy, and a genetic study using a gene panel test, looking for mutations in the PIKFYVE gene. RESULTS: A slit-lamp examination of the first family revealed polychromatic crystalline punctiform opacities distributed all over the stroma in 8 of 11 family members in three generations with an autosomal dominant inheritance. The second family showed in three of four members in two generations the same opacities located in the pre-Descemet region. It was also a hint for autosomal dominant inheritance. The in vivo confocal microscopy identified numerous rounded and hyperreflective stromal particles measuring 10-15 µm in diameter, with the highest density in the posterior stroma and with normal keratocytes. No systemic disease was diagnosed. No variants or mutations were identified in PIKFYVE gene. CONCLUSIONS: Polychromatic deposits in patients with Punctiform and Polychromatic Pre-Descemet corneal dystrophy can be located not only in the deep stroma but also in the anterior and middle stroma. Our presentation reveals the possibility of considering this characteristic corneal disorder as a corneal dystrophy of its own and not as a subtype of pre-Descemet corneal dystrophy.

Schnyder corneal dystrophy and associated phenotypes caused by novel and recurrent mutations in the UBIAD1 gene.

BACKGROUND: The purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands. METHODS: We identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family. RESULTS: A novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested. CONCLUSION: Although de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.

Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients.

Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor ß-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor ß-induced protein (amino acid 611-642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.