Wild-type Lenses Research Articles

Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

Disruption of Eph-ephrin bidirectional signaling leads to human congenital and age-related cataracts, but the mechanisms for these opacities in the eye lens remain unclear. Eph receptors bind to ephrin ligands on neighboring cells to induce canonical ligand-mediated signaling. The EphA2 receptor also signals non-canonically without ligand binding in cancerous cells, leading to epithelial-to-mesenchymal transition (EMT). We have previously shown that the receptor EphA2 and the ligand ephrin-A5 have diverse functions in maintaining lens transparency in mice. Loss of ephrin-A5 leads to anterior cataracts due to EMT. Surprisingly, both canonical and non-canonical EphA2 activation are present in normal wild-type lenses and in the ephrin-A5 knockout lenses. Canonical EphA2 signaling is localized exclusively to lens epithelial cells and does not change with age. Non-canonical EphA2 signaling is in both epithelial and fiber cells and increases significantly with age. We hypothesize that canonical ligand-dependent EphA2 signaling is required for the morphogenesis and organization of hexagonal equatorial epithelial cells while non-canonical ligand-independent EphA2 signaling is needed for complex membrane interdigitations that change during fiber cell differentiation and maturation. This is the first demonstration of non-canonical EphA2 activation in a non-cancerous tissue or cell and suggests a possible physiological function for ligand-independent EphA2 signaling.

TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton.

Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses.

Dysregulation of Autophagy Occurs During Congenital Cataract Development in βA3ΔG91 Mice.

To examine lens phenotypic characteristics in βA3ΔG91 mice and determine if βA3ΔG91 affects autophagy in the lens. We generated a βA3ΔG91 mouse model using CRISPR/Cas9 methodology. Comparative phenotypic and biochemical characterizations of lenses from postnatal day 0 (P0), P15, and 1-month-old βA3ΔG91 and wild-type (WT) mice were performed. The methodologies used included non-invasive slit-lamp examination, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemical (IHC) analyses to determine the levels of autophagy-related genes and proteins. Transmission electron microscopy (TEM) analysis of lenses was performed to assess organelle degradation and the presence of autophagic vesicles. TUNEL staining was used to determine apoptosis in the lens. Relative to WT lenses, 1-month-old βA3ΔG91 mice developed congenital nuclear cataract and microphthalmia and showed an early loss of endoplasmic reticulum (ER) in the cortex and attenuation of nuclei degradation. This observation was confirmed by TEM analysis, as was the presence of autophagic vesicles in βA3ΔG91 lenses. Comparative IHC and RT-qPCR analyses showed relatively higher levels of autophagy markers (ubiquitinated proteins and p62, LC3, and LAMP2 proteins) in βA3ΔG91 lenses compared to WT lenses. Additionally, βA3ΔG91 lenses showed relatively greater numbers of apoptotic cells and higher levels of cleaved caspase-3 and caspase-9. The deletion of G91 in βA3ΔG91 mice leads to higher levels of expression of autophagy-related proteins and their transcripts relative to WT lenses. Taken together, G91 deletion in βA3/A1-crystallin is associated with autophagy disruption, attenuation of nuclei degradation, and cellular apoptosis in the lens, which might be congenital cataract causative factors.

A crystallin mutant cataract with mineral deposits

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increasedlevels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.

Connexin Mutants Cause Cataracts Through Deposition of Apatite.

Cataracts are lens opacities that are among the most common causes of blindness. It is commonly believed that cataracts develop through the accumulation of damage to lens proteins. However, recent evidence suggests that cataracts can result from calcium ion accumulation and the precipitation of calcium-containing salts. To test for the presence of precipitates and to identify their components, we studied the lenses of mice that develop cataracts due to mutations of connexin46 and connexin50. Micro-computed tomography showed the presence of radio-dense mineral in the mutant lenses, but not in wild-type lenses. Three-dimensional reconstructions of the scans showed that the distribution of the radio-dense mineral closely paralleled the location and morphology of the cataracts. The mutant lens homogenates also contained insoluble particles that stained with Alizarin red (a dye that stains Ca2+ deposits). Using attenuated total internal reflection micro–Fourier transform infrared spectroscopy, we identified the mineral as calcium phosphate in the form of apatite. Taken together, these data support the novel paradigm that cataracts are formed through pathological mineralization within the lens.

Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4−/− mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-β-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

Transforming growth factor-β2-mediated mesenchymal transition in lens epithelial cells is repressed in the absence of RAGE

Transforming growth factor-β2 (TGFβ2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFβ2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFβ2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFβ2-mediated Smad signaling. In addition, treatment with TGFβ2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFβ2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin β1 in capsule-adherent LECs. Taken together, these results suggest that TGFβ2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

Studies on the Involvement of Integrin and the Tubulin Cytoskeleton in the Mechanism Responsible for TRPV1‐dependent NKCC1 Activation in Mouse Lens

Previously we showed shrinkage of the mouse lens by hyperosmotic solution causes TRPV1-dependent activation of NKCC1. In various tissues the cytoskeleton has been shown to play a role in responses to osmotic stress. Here we present evidence for the involvement of integrin and tubulin in the activation of NKCC1 in mouse lens epithelium. Studies were carried out on lenses from wild-type (WT) and TRPV1 knockout (KO) mice. Rubidium (Rb) uptake, measured using an atomic absorption spectrophotometer, was used as a measure of NKCC activity. ERK1/2 phosphorylation was measured by quantitative Western blot analysis. Intracellular calcium was measured in Fura-2 loaded cultured lens epithelium by a ratiometric imaging technique. In WT mice, the integrin agonist leukadherin 1 (LA-1, 25 μM) increased Rb uptake by 33% in intact lenses and increased intracellular calcium concentration in cultured lens epithelium from 197±18 to 341±25 nM in (n=5, P<0.001). In contrast, LA-1 failed to produce any significant change of Rb uptake in TRPV1 KO lenses and did not increase calcium in cultured lens epithelium from TRPV1 KO animals. The increase of Rb uptake elicited by LA-1 in WT lenses was abolished by the putative tubulin stabilizer, paclitaxel (100 nm). The TRPV1 antagonist, A889425 (1.0 μM) also abolished the Rb uptake response to LA-1. LA-1, the TRPV1 agonist capsaicin, and hyperosmotic solution (350 mOsm) all caused ERK1/2 activation in WT cultured lens epithelium. In each case, the ERK1/2 response was transient with a peak response at ~5 min. The ERK1/2 activation responses to hyperosmotic stress and LA-1 induced were markedly suppressed by A889425 and by paclitaxel. Taken together, the results point to a link between integrin, the tubulin cytoskeleton, and TRPV1-dependent activation of NKCC1 in the mouse lens. It is noteworthy that the integrin agonist LA-1 as well as hyperosmotic stress both were found to activate ERK1/2, the upstream kinase that was shown previously associated with activation of NKCC1 in the lens.

Biochemical and biomechanical characteristics of dystrophin-deficient mdx3cv mouse lens

The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx3cv mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx3cv mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx3cv mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx3cv mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx3cv mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx3cv lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.

The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology.

Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1-/- and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1-/- lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1-/- cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (<2 min) NKCC1 phosphorylation in WT but not TRPV1-/- cells. HP recovery was shown to be absent in TRPV1-/- lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1-/- mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

Connexin 50 and AQP0 are Essential in Maintaining Organization and Integrity of Lens Fibers.

PurposeConnexins and aquaporins play essential roles in maintaining lens homeostasis and transparency and there is a close physical and functional relationship between these two proteins. Aquaporin 0 (AQP0), in addition to its role in water transport in the lens, acts as a cell–cell adhesion molecule. Recently, we showed a new role of connexin (Cx) 50 in mediating cell–cell adhesion. However, the cooperative roles of these two proteins in the lens in vivo have not been reported.MethodsWe generated an AQP0/Cx50 double knockout (dKO) mouse model. Light, fluorescence, transmission thin section, and freeze-fracture electron microscopy, as well as wheat germ agglutinin and phalloidin labeling were used to evaluate lens structure. Mechanical properties of lenses were determined by mechanical compression testing.ResultsDKO mice exhibited small eyes and lenses with severe cataracts, along with lens posterior defects, including posterior capsule rupture. The dKO mouse lenses had severe structural disruption associated with increased spaces between lens fiber cells when compared with wild-type lenses or lenses deficient in either Cx50 or AQP0. DKO mice also exhibited greater reduction in lens size compared with Cx50 KO mice. Gap-junction plaque size was greatly decreased in cortical fiber cells in dKO mice. Moreover, lens stiffness and elasticity were completely diminished, exhibiting a gelatinous texture in adult dKO mice.ConclusionsThis novel mouse model reveals that Cx50 and AQP0 play an important role in mediating cell–cell adhesion function in the lens fiber cells and their deficiency impairs lens fiber organization, integrity, mechanical properties, and lens development.

C-Terminal End of Aquaporin 0 Regulates Lens Gap Junction Channel Function.

PurposeWe reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation.MethodsA mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels.ResultsAt postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50.ConclusionsThe increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.

The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation.

PurposeMutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation.MethodsConnexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+]i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images.ResultsIn Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+]i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts.ConclusionsCx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken βB1-crystallin promoter was used to overexpress Loxl1 in the lenses of βB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. βB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20–50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of βB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2–5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

Bioinformatics analysis of microarray data to explore the key genes involved in HSF4 mutation-induced cataract.

To reveal the mechanisms of heat-shock transcription factor 4 (HSF4) mutation-induced cataract. GSE22362, including 3 HSF4-null lens and 3 wild-type lens, was obtained from Gene Expression Omnibus database. After data preprocessing, the differentially expressed genes (DEGs) were identified using the limma package. Based on Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, functional and pathway enrichment analyses were performed for the DEGs. Followed by protein-protein interaction (PPI) network was constructed using STRING database and Cytoscape software. Furthermore, the validated microRNA (miRNA)-DEG pairs were obtained from miRWalk2.0 database, and then miRNA-DEG regulatory network was visualized by Cytoscape software. A total of 176 DEGs were identified in HSF4-null lens compared with wild-type lens. In the PPI network, FBJ osteosarcoma oncogene (FOS), early growth response 1 (EGR1) and heme oxygenase (decycling) 1 (HMOX1) had higher degrees and could interact with each other. Besides, mmu-miR-15a-5p and mmu-miR-26a-5p were among the top 10 miRNAs in the miRNA-DEG regulatory network. Additionally, mmu-miR-26a-5p could target EGR1 in the regulatory network. FOS, EGR1, HMOX1, mmu-miR-26a-5p and mmu-miR-15a-5p might function in the pathogenesis of HSF4 mutation-induced cataract.

Aqp0a Regulates Suture Stability in the Zebrafish Lens.

PurposeTo investigate the roles of Aquaporin 0a (Aqp0a) and Aqp0b in zebrafish lens development and transparency.MethodsCRISPR/Cas9 gene editing was used to generate loss-of-function deletions in zebrafish aqp0a and/or aqp0b. Wild type (WT), single mutant, and double mutant lenses were analyzed from embryonic to adult stages. Lens transparency, morphology, and growth were assessed. Immunohistochemistry was used to map protein localization as well as to assess tissue organization and distribution of cell nuclei.Resultsaqp0a−/− and/or aqp0b−/− cause embryonic cataracts with variable penetrance. While lenses of single mutants of either gene recover transparency in juveniles, double mutants consistently form dense cataracts that persist in adults, indicating partially redundant functions. Double mutants also reveal redundant Aqp0 functions in lens growth. The nucleus of WT lenses moves from the anterior pole to the lens center with age. In aqp0a−/− mutants, the nucleus fails to centralize as it does in WT or aqp0b−/− lenses, and in double mutant lenses there is no consistent lens nuclear position. In addition, the anterior sutures of aqp0a−/−, but not aqp0b−/− mutants, are unstable resulting in failure of suture maintenance at older stages and anterior polar opacity.Conclusions. Zebrafish Aqp0s have partially redundant functions, but only Aqp0a promotes suture stability, which directs the lens nucleus to centralize, failure of which results in anterior polar opacity. These studies support the hypothesis that the two Aqp0s subfunctionalized during fish evolution and that Aqp0-dependent maintenance of the anterior suture is essential for lens transparency.

Inverse elastographic method for analyzing the ocular lens compression test

The ocular lens stiffens dramatically with age, resulting in a loss of function. However, the mechanism of stiffening remains unknown, at least in part due to difficulties in making reliable measurements of the intrinsic mechanical properties of the lens. Recent experiments have employed manual compression testing to evaluate the stiffness of murine lenses which have genotypes pertinent to human lens diseases. These experiments compare the extrinsic stiffness of lenses from the genotype of interest to the wild-type lens in an effort to reach conclusions regarding the cellular or molecular basis of lens stiffening. However, these comparisons are confounded by alterations in lens size and geometry which invariably accompany these genetic manipulations. Here, we utilize manual lens compression to characterize the stiffness of a porcine lens and a murine lens. An inverse elastographic technique was then developed to estimate the intrinsic shear modulus of each lens as well as the elastic modulus of the lens capsule. The results were in good agreement with the previous literature values.

Abstract 4638: The p53 suppression on tumor cells may recruit macrophages and modulate their expression of the indoleamine 2, 3-dioxygenase

Abstract We had found that expression of indoleamine 2,3-dioxygenase (IDO) on cervical cancer cells increased as it progressed from carcinoma in situ (CIS) to microinvasive cancer, and that tumor-associated macrophages (TAM) recruited at the invasive front also expressed IDO in cervical cancer patients (Cancer Sci 98:874-881 2007). We previously produced homozygous αT3 transgenic mice carrying two copies of αA-SV40Tag transgene (αA denotes murine αA-crystallin promoter) in order to study DNA virus-induced carcinogenesis. The αT3 mice developed undifferentiated cancer of lens epithelium, which tumors showed multi-step carcinogenesis which was similar to cervical cancer (J Cancer Res Clin Oncol 135:1521-1532 2009). We found that homozygous αT3 mice carrying two copies of SV40Tag transgene progressed faster and died earlier than hemizygous αT3 mice carrying one copy of Tag transgene. In this study, in order to examine the relationship between p53 function and tumor progression, αT3 mice were mated with M1 mice expressing mutant p53 in lens cells (Proc. Natl. Acad. Sci. USA, 92:6142-6146, 1995) producing αT3M1 mice, and also were mated with p53-deficient mice to produce p53-deficient αT3 (αT3p53(-/-)) mice( Cancer Letters 179:165-173,2002, Oncology Report 12:253-258, 2004). At 20 weeks of age, the proportion of progression from CIS to invasive cancer was analyzed by H&amp;E staining and the number of recruited TAM and IDO-expressing TAM were determined by immunohistochemical staining and FACS analysis. Pathologically, both αT3M1 and αT3p53(-/-) lens tumors significantly progressed to invasive cancer more than αT3 tumors. There were a few macrophages in wild-type lens, but more TAM were recruited in both αT3, αT3M1 and αT3p53(-/-) lens tumors. However, no significant difference of the number of IDO-expressing macrophages in peritoneal cavity was detected in each mice of wild-type, αT3, αT3M1 and αT3p53(-/-). It suggested that p53 suppression on tumor cells might modulate IDO-expression for macrophages. Moreover, we established the tumor cell lines from each αT3, αT3M1 and αT3p53(-/-) lens tumors. The peritoneal macrophages were analyzed for IDO-expression in 4 days after i.p. injection of these cells. Significantly, IDO-expressing macrophages in peritoneal cavity were more detected after i.p. injection of each αT3M1 and αT3p53(-/-) cells than i.p. injection of αT3 cells. And the co-culture of peritoneal macrophages with each αT3M1 and αT3p53(-/-) tumor cells also induced the IDO-expression of macrophages more than the co-culture with αT3 cells. These results suggested that the amount of p53 suppression on tumor cells would be related to the number of recruited TAM and their expression of IDO. The p53 suppression on tumor cells may recruit TAM and affect the macrophage function and, suppress an innate immunity possibly through the expression of IDO, which may enhance invasion and progression of tumors. Citation Format: Takafumi Nakamura. The p53 suppression on tumor cells may recruit macrophages and modulate their expression of the indoleamine 2, 3-dioxygenase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4638. doi:10.1158/1538-7445.AM2017-4638