Alexander I. Son

Loss of ephrin-A5 function disrupts lens fiber cell packing and leads to cataract

Cell–cell interactions organize lens fiber cells into highly ordered structures to maintain transparency. However, signals regulating such interactions have not been well characterized. We report here that ephrin-A5, a ligand of the Eph receptor tyrosine kinases, plays a key role in lens fiber cell shape and cell–cell interactions. Lens fiber cells in mice lacking ephrin-A5 function appear rounded and irregular in cross-section, in contrast to their normal hexagonal appearance in WT lenses. Cataracts eventually develop in 87% of ephrin-A5 KO mice. We further demonstrate that ephrin-A5 interacts with the EphA2 receptor to regulate the adherens junction complex by enhancing recruitment of β-catenin to N-cadherin. These results indicate that the Eph receptors and their ligands are critical regulators of lens development and maintenance.

Human cataract mutations in EPHA2 SAM domain alter receptor stability and function.

The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity.

Roles of EphA2 in Development and Disease

The Eph family of receptor tyrosine kinases (RTKs) has been implicated in the regulation of many aspects of mammalian development. Recent analyses have revealed that the EphA2 receptor is a key modulator for a wide variety of cellular functions. This review focuses on the roles of EphA2 in both development and disease.

The role of Eph receptors in lens function and disease.

Cataract is the single largest contributor to blindness in the world, with the disease having a strong genetic component. In recent years the Eph family of receptor tyrosine kinases has been identified as a key regulator in lens clarity. In this review we discuss the roles of the Eph receptors in lens biology and cataract development.

Formation of persistent hyperplastic primary vitreous in ephrin-A5-/- mice.

PURPOSE Primary vitreous regression is a critical event in mammalian eye development required for proper ocular maturity and unhindered vision. Failure of this event results in the eye disease persistent hyperplastic primary vitreous (PHPV), also identified as persistent fetal vasculature (PFV), a condition characterized by the presence of a fibrovascular mass adjacent to the lens and retina, and associated with visual disability and blindness. Here, we identify ephrin-A5 to be a critical regulator for primary vitreous regression. METHODS Wild-type and ephrin-A5(-/-) eyes were examined at various developmental stages to determine the progression of PHPV. Eye tissue was sectioned and examined by H&E staining. Protein expression and localization was determined through immunohistochemistry. Relative levels of Eph receptors were determined by RT-PCR. RESULTS Ephrin-A5(-/-) animals develop ocular phenotypes representative of PHPV, most notably the presence of a large hyperplastic mass posterior to the lens that remains throughout the lifetime of the animal. The aberrant tissue in these mutant mice consists of residual hyaloid vessels surrounded by pigmented cells of neural crest origin. Labeling with bromodeoxyuridine (BrdU) and detection of proliferating cell nuclear antigen (PCNA) expression shows that the mass in ephrin-A5(-/-) animals is mitotically active in embryonic and postnatal stages. CONCLUSIONS Ephrin-A5 is a critical factor that regulates primary vitreous regression.

Growth Cone Collapse Assay

Growth cone collapse is an easy and efficient test for detecting and characterizing axon guidance activities secreted or expressed by cells. It can also be used to dissect signaling pathways by axon growth inhibitors and to isolate therapeutic compounds that promote axon regeneration. Here, we describe a growth cone collapse assay protocol used to study signal transduction mechanisms of the repulsive axon guidance molecule ephrin-A5 in hippocampal neurons.

Ephrin-A5 Is Required for Optimal Fertility and a Complete Ovulatory Response to Gonadotropins in the Female Mouse

Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.