Chun-hong Xia

Connexins in Lens Development and Cataractogenesis

The lens is an avascular organ that transmits and focuses light images onto the retina. Intercellular gap junction channels, formed by at least three different connexin protein subunits, α1 (connexin43 or Gja1), α3 (connexin46 or Gja3) and α8 (connexin50 or Gja8), are utilized to transport metabolites, ions and water in the lens. In combination with physiological and biochemical analyses, recent genetic studies have significantly improved our understanding about the roles of diverse gap junction channels formed by α3 and α8 connexin subunits during lens development and cataract formation. These studies have demonstrated that α3 connexin is essential for lens transparency while α8 connexin is important for lens growth and transparency. Diverse gap junction channels formed by α3 and α8 subunits are important for the differentiation, elongation and maturation of lens fiber cells. Aberrant gap junction communication, caused by alterations of channel assembly, channel gating or channel conductance, can lead to different types of cataracts. These findings provide some molecular insights for essential roles of connexins and gap junctions in lens formation and the establishment and maintenance of lifelong lens transparency.

Knock-in of α3 connexin prevents severe cataracts caused by an α8 point mutation

A G22R point mutation in α8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type α3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in α3 connexin expressed on the locus of the endogenous α8 connexin could modulate the severe cataract caused by the α8-G22R mutation. We found that the α3(-/-) α8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the α3(-/-) α8(G22R/KIα3) lens contained relatively normal inner fibers without lens posterior rupture. The α8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of α3(-/-) α8(G22R/KIα3) lenses, but not in those of α3(-/-) α8(G22R/-) lenses. Thus, we hypothesize that the knocked-in α3 connexin subunits interact with the α8-G22R connexin subunits to form functional gap junction channels and rescue the lens phenotype. Using an electrical coupling assay consisting of paired Xenopus oocytes, we demonstrated that only co-expression of mutant α8-G22R and wild-type α3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by α3 connexin subunits alone. Thus, knocked-in α3 connexin and mutant α8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency.

The cataract-inducing S50P mutation in Cx50 dominantly alters the channel gating of wild-type lens connexins

Mutations within connexin50 (Cx50) have been linked to various cataract phenotypes. To determine the mechanism behind cataract formation we used the paired Xenopus oocyte system in conjunction with transfected HeLa cells and genetically engineered mouse models to examine the functional characteristics of gap junctions in which a cataract-causing mutant of Cx50 (hereafter referred to as Cx50-S50P) is expressed. Channels comprising Cx50-S50P subunits alone failed to induce electrical coupling. However, the mixed expression of Cx50-S50P and wild-type subunits of either Cx50 or Cx46 – to create heteromeric gap junctions – resulted in functional intercellular channels with altered voltage-gating properties compared with homotypic wild-type channels. Additionally, immunofluorescence microscopy showed that channels of Cx50-S50P subunits alone failed to localize to the plasma membrane – unlike channels composed of Cx46 subunits, which concentrated at cell-cell appositions. Cx50-S50P colocalized with wild-type Cx46 in both transfected HeLa cells in vitro and mouse lens sections in vivo. Taken together, these data define the electrophysiological properties and intracellular targeting of gap junctions formed by the heteromeric combination of Cx50 or Cx46 and Cx50-S50P mutant proteins. Additionally, mixed channels displayed significantly altered gating properties, a phenomenon that may contribute to the cataract that is associated with this mutation.

Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one alleles function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice.

Dense Nuclear Cataract Caused by the γB-Crystallin S11R Point Mutation

PURPOSE To identify the causative gene mutation for a new dominant cataract in mice and to investigate the molecular basis for how the mutated gene leads to a dense nuclear cataract. METHODS Genomewide linkage analysis and DNA sequencing were used to determine the gene mutation. Histology, immunohistochemistry, and Western blotting were used to characterize lens phenotypes. Ion concentrations were measured by an inductively coupled plasma-optical emission spectrometer (ICP-OES). RESULTS A point mutation (A to C) of the gammaB-crystallin gene, which results in the gammaB-S11R mutant protein, was identified in this cataractous mouse line. Homozygous mutant mice developed dense nuclear cataracts associated with disrupted inner lens fiber cells. Immunohistochemistry data revealed gamma-crystallin aggregates at the cell boundaries of inner mature fibers that lose actin filaments. Western blotting showed an increased degradation of crystallin proteins correlated with the nuclear cataract. ICP-OES confirmed a substantial elevation of calcium concentration in mutant lenses. CONCLUSIONS This dominant cataract was caused by the gammaB-S11R mutation. Mutant gammaB-S11R proteins triggered the gamma-crystallin aggregation that probably disrupted membrane-cytoskeleton structures of inner fiber cells, causing increased calcium influxes. Subsequent activation of calcium-dependent protein degradation and degeneration of inner mature fiber cells led to the dense nuclear cataract.

Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8R205G mutation occurs at the same conserved residue as the human GJA8R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.

Connexin Mediated Cataract Prevention in Mice

Cataracts, named for any opacity in the ocular lens, remain the leading cause of vision loss in the world. Non-surgical methods for cataract prevention are still elusive. We have genetically tested whether enhanced lens gap junction communication, provided by increased α3 connexin (Cx46) proteins expressed from α8(Kiα3) knock-in alleles in Gja8tm1(Gja3)Tww mice, could prevent nuclear cataracts caused by the γB-crystallin S11R mutation in CrygbS11R/S11R mice. Remarkably, homozygous knock-in α8(Kiα3/Kiα3) mice fully prevented nuclear cataracts, while single knock-in α8(Kiα3/−) allele mice showed variable suppression of nuclear opacities in CrygbS11R/S11R mutant mice. Cataract prevention was correlated with the suppression of many pathological processes, including crystallin degradation and fiber cell degeneration, as well as preservation of normal calcium levels and stable actin filaments in the lens. This work demonstrates that enhanced intercellular gap junction communication can effectively prevent or delay nuclear cataract formation and suggests that small metabolites transported through gap junction channels protect the stability of crystallin proteins and the cytoskeletal structures in the lens core. Thus, the use of an array of small molecules to promote lens homeostasis may become a feasible non-surgical approach for nuclear cataract prevention in the future.

Gap junction communication influences intercellular protein distribution in the lens

Lens transparency and high refractive index presumably depend on the appropriate arrangement and distribution of lens proteins among lens fiber cells. Intercellular gap junction channels formed by alpha3 and alpha8 connexins are known to transport small molecules, ions and water, but not proteins, in the lens. Mosaic expression of green fluorescent protein (GFP) in the lens is a useful marker for monitoring macromolecule distribution between fiber cells and for constructing three-dimensional images of living lens cells. In alpha3(-/-) alpha8(-/-) double knockout (DKO) lenses, three-dimensional images of GFP-positive cells demonstrate the changes of epithelial cell surfaces and insufficient elongation of inner fiber cells. Uniform distribution of GFP between inner lens fiber cells is observed in both wild-type and alpha3(-/-) lenses. In contrast, uniform GFP distribution is slightly delayed in alpha8(-/-) lenses and is abolished in DKO lenses. Without endogenous wild-type alpha3 and alpha8 connexins, knock-in alpha3 connexin (expressed under the alpha8 gene promoter) restores the uniform distribution of GFP protein in the lens. Thus, the presence of either alpha3 or alpha8 connexins seems sufficient to support the uniform distribution of GFP between differentiated lens fiber cells. Although the mechanism that drives GFP transport between fiber cells remains unknown, this work reveals that gap junction communication plays a novel role in the regulation of intercellular protein distribution in the lens.

Review article screening, genetics, risk factors, and treatment of neonatal cataracts

Neonatal cataracts remain the most common cause of visual loss in children worldwide and have diverse, often unknown, etiologies. This review summarizes current knowledge about the detection, treatment, genetics, risk factors, and molecular mechanisms of congenital cataracts. We emphasize significant progress and topics requiring further study in both clinical cataract therapy and basic lens research. Advances in genetic screening and surgical technologies have improved the diagnosis, management, and visual outcomes of affected children. For example, mutations in lens crystallins and membrane/cytoskeletal components that commonly underlie genetically inherited cataracts are now known. However, many questions still remain regarding the causes, progression, and pathology of neonatal cataracts. Further investigations are also required to improve diagnostic criteria for determining the timing of appropriate interventions, such as the implantation of intraocular lenses and postoperative management strategies, to ensure safety and predictable visual outcomes for children. Birth Defects Research 109:734–743, 2017.

NHE8 Is Essential for RPE Cell Polarity and Photoreceptor Survival

A new N-ethyl-N-nitrosourea (ENU)-induced mouse recessive mutation, identified by fundus examination of the eye, develops depigmented patches, indicating retinal disorder. Histology data show aberrant retinal pigment epithelium (RPE) and late-onset photoreceptor cell loss in the mutant retina. Chromosomal mapping and DNA sequencing reveal a point mutation (T to A) of the Slc9a8 gene, resulting in mutant sodium/proton exchanger 8 (NHE8)-M120K protein. The lysine substitution decreases the probability of forming the 3rd transmembrane helix, which impairs the pore structure of the Na+/H+ exchanger. Various RPE defects, including mislocalization of the apical marker ezrin, and disrupted apical microvilli and basal infoldings are observed in mutant mice. We have further generated NHE8 knockout mice and confirmed similar phenotypes, including abnormal RPE cells and late-onset photoreceptor cell loss. Both in vivo and in vitro data indicate that NHE8 co-localizes with ER, Golgi and intracellular vesicles in RPE cells. Thus, NHE8 function is necessary for the survival of photoreceptor cells and NHE8 is important for RPE cell polarity and function. Dysfunctional RPE may ultimately lead to photoreceptor cell death in the NHE8 mutants. Further studies will be needed to elucidate whether or not NHE8 regulates pH homeostasis in the protein secretory pathways of RPE.