Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal Dystrophies.

The cornea, the eye's outermost layer, protects the eye from the environment. The cornea's innermost layer is an endothelium separating the stromal layer from the aqueous humor. A central role of the endothelium is to maintain stromal hydration state. Defects in maintaining this hydration can impair corneal clarity and thus visual acuity. Two endothelial corneal dystrophies, Fuchs Endothelial Corneal Dystrophy (FECD) and Congenital Hereditary Endothelial Dystrophy (CHED), are blinding corneal diseases with varied clinical presentation in patients across different age demographics. Recessive CHED with an early onset (typically age: 0-3 years) and dominantly inherited FECD with a late onset (age: 40-50 years) have similar phenotypes, although caused by defects in several different genes. A range of molecular mechanisms have been proposed to explain FECD and CHED pathology given the involvement of multiple causative genes. This critical review provides insight into the proposed molecular mechanisms underlying FECD and CHED pathology along with common pathways that may explain the link between the defective gene products and provide a new perspective to view these genetic blinding diseases.

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.

Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies.

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Multi-pronged proteomic analysis to study the glioma pathobiology using cerebrospinal fluid samples.

PURPOSE: Gliomas are one of the most aggressive and lethal brain tumors arising from neoplastic transformation of astrocytes and oligodendrocytes. A comprehensive quantitative analysis of proteome level differences in cerebrospinal fluid (CSF) across different grades of gliomas for a better understanding of glioma pathobiology is carried out. EXPERIMENTAL DESIGN: Glioma patients are diagnosed by radiology and histochemistry-based analyses. Differential proteomic analysis of high (n = 12) and low (n = 5) grade gliomas, and control (n = 3) samples is performed by using two complementary quantitative proteomic approaches; 2D-DIGE and iTRAQ. Further, comparative analysis of three IDH wild-type and five IDH mutants is performed to identify the proteome level differences between these two sub-classes. RESULTS: Level of several proteins including haptoglobin, transthyretin, osteopontin, vitronectin, complement factor H and different classes of immunoglobulins are found to be considerably increased in CSF of higher grades of gliomas. Subsequent bioinformatics analysis indicated that many of the dysregulated CSF proteins are associated with metabolism of lipids and lipoproteins, complement and coagulation cascades and extracellular matrix remodeling in gliomas. Intriguingly, CSF of glioma patients with IDH mutations exhibite increased levels of multiple proteins involved in response to oxidative stress. CONCLUSION AND CLINICAL RELEVANCE: To the best of our knowledge, this is the foremost proteome level investigation describing comprehensive proteome profiles of different grades of gliomas using proximal fluid (CSF); and thereby providing insights into disease pathobiology, which aided in identification of grade and sub-type specific alterations. Moreover, if validated in larger clinical cohorts, a panel of differentially abundant CSF proteins may serve as potential disease monitoring and prognostic markers for gliomas.

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction.

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.