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 3(rd) 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.

A model for familial exudative vitreoretinopathy caused by LPR5 mutations.

We have identified a mouse recessive mutation that leads to attenuated and hyperpermeable retinal vessels, recapitulating some pathological features of familial exudative vitreoretinopathy (FEVR) in human patients. DNA sequencing reveals a single nucleotide insertion in the gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5), causing a frame shift and resulting in the replacement of the C-terminal 39 amino acid residues by 20 new amino acids. This change eliminates the last three PPP(S/T)P repeats in the LRP5 cytoplasmic domain that are important for mediating Wnt/beta-catenin signaling. Thus, mutant LRP5 protein is probably unable to mediate its downstream signaling. Immunostaining and three-dimensional reconstructions of retinal vasculature confirm attenuated retinal vessels. Ultrastructural data further reveal that some capillaries lack lumen structure in the mutant retina. We have also verified that LRP5 null mice develop similar alterations in the retinal vasculature. This study provides direct evidence that LRP5 is essential for the development of retinal vasculature, and suggests a novel role played by LRP5 in capillary maturation. LRP5 mutant mice can be a useful model to explore the clinical manifestations of FEVR.

Arginine 54 and Tyrosine 118 residues of {alpha}A-crystallin are crucial for lens formation and transparency.

PURPOSE: To identify new mouse models for studying roles of alphaAlpha-crystallin in vivo and to investigate why and how different mutations of the alphaAlpha-crystallin gene lead to dominant or recessive cataracts. METHODS: Using mouse genetic approaches and slit lamp screening, we identified two mouse cataractous mutant lines. Causative genes were mapped by a genome-wide linkage analysis. DNA sequencing verified missense mutations of alphaA-crystallin gene in both mutant lines. Histology, imaging of green fluorescent protein (GFP)-positive lenses, and protein 2-DE gel were used to determine the morphologic and biochemical properties of mutant lenses. RESULTS: Two new alphaA-crystallin gene mutations were identified, alphaA-R54C (alphaA-Cys) and alphaA-Y118D, which cause recessive whole cataracts and dominant nuclear cataracts, respectively. In homozygous alphaA-Cys mutant mice, lens epithelial and fiber cells lost their characteristic cellular features and developed disrupted subcellular structures, such as actin filaments and mitochondria. The nuclear cataract caused by alphaA-Y118D mutation was associated with increased water-insoluble crystallins (alpha, beta, and gamma classes). These results suggest that the Arg54 residue in the N-terminal region is crucial for alphaA-crystallin to perform its roles in lens epithelial and fiber cells during development, whereas the Y118D mutation in the central alpha-crystallin domain impairs alphaA-crystallin's ability to maintain the solubility of crystallin proteins in the lens. CONCLUSIONS: This work demonstrates that different regions of alphaA-crystallin mediate distinct functions in vivo. These two mutant mouse lines provide useful animal models for further investigating the multiple roles of alphaA-crystallin in the lens.

Absence of alpha3 (Cx46) and alpha8 (Cx50) connexins leads to cataracts by affecting lens inner fiber cells.

Lens development and transparency have been hypothesized to depend on intercellular gap junction channels, consisting of alpha3 (Cx46) and alpha8 (Cx50) connexin subunits, to transport metabolites, secondary messages and ions between lens cells. To evaluate this hypothesis, we have generated alpha3(-/-) alpha8(-/-) double knockout mice and characterized their lens phenotypes. Without gap junctions between lens fiber cells, alpha3(-/-) alpha8(-/-) lenses displayed severe cataracts resulting from cell swelling and degeneration of inner fibers while normal peripheral fiber cells continued to form throughout life. Neither an increase of degraded crystallins nor an increase of water-insoluble crystallins was found in alpha3(-/-) alpha8(-/-) lenses. However, a substantial reduction of gamma-crystallin proteins, but not alpha- and beta-crystallins, was detected. These results suggest that gap junction communication is important for maintaining lens homeostasis of inner fiber cells and that a loss of gap junctions leads to cataract formation as well as reductions of gamma-crystallin proteins and transcripts.

Knock-in of alpha3 connexin prevents severe cataracts caused by an alpha8 point mutation.

A G22R point mutation in alpha8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type alpha3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in alpha3 connexin expressed on the locus of the endogenous alpha8 connexin could modulate the severe cataract caused by the alpha8-G22R mutation. We found that the alpha3(-/-) alpha8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the alpha3(-/-) alpha8(G22R/KIalpha3) lens contained relatively normal inner fibers without lens posterior rupture. The alpha8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of alpha3(-/-) alpha8(G22R/KIalpha3) lenses, but not in those of alpha3(-/-) alpha8(G22R/-) lenses. Thus, we hypothesize that the knocked-in alpha3 connexin subunits interact with the alpha8-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 alpha8-G22R and wild-type alpha3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by alpha3 connexin subunits alone. Thus, knocked-in alpha3 connexin and mutant alpha8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency.

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis.

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.