Mir-204-5p alleviates mitochondrial dysfunction by targeting IGFBP5 in diabetic cataract.

BACKGROUND: Cataract contributes to visual impairment worldwide, and diabetes mellitus accelerates the formation and progression of cataract. Here we found that the expression level of miR-204-5p was diminished in the lens epithelium with anterior lens capsule of cataract patients compared to normal donors, and decreased more obviously in those of diabetic cataract (DC) patients. However, the contribution and mechanism of miR-204-5p during DC development remain elusive. METHODS AND RESULT: The mitochondrial membrane potential (MMP) was reduced in the lens epithelium with anterior lens capsule of DC patients and the H2O2-induced human lens epithelial cell (HLEC) cataract model, suggesting impaired mitochondrial functional capacity. Consistently, miR-204-5p knockdown by the specific inhibitor also attenuated the MMP in HLECs. Using bioinformatics and a luciferase assay, further by immunofluorescence staining and Western blot, we identified IGFBP5, an insulin-like growth factor binding protein, as a direct target of miR-204-5p in HLECs. IGFBP5 expression was upregulated in the lens epithelium with anterior lens capsule of DC patients and in the HLEC cataract model, and IGFBP5 knockdown could reverse the mitochondrial dysfunction in the HLEC cataract model. CONCLUSIONS: Our results demonstrate that miR-204-5p maintains mitochondrial functional integrity through repressing IGFBP5, and reveal IGFBP5 may be a new therapeutic target and prognostic factor for DC.

The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and ßγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the ßγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens ßγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in ßB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even ß-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Macrophages modulate fibrosis during newt lens regeneration.

BACKGROUND: Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated. METHODS: Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery. RESULTS: Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process. CONCLUSIONS: Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens.

The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant ßB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.

Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults.

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H2O2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress.

Characterization of mechanical stress in the occurrence of cortical opacification in age-related cataracts using three-dimensional finite element model of the human lens and RNA-seq.

Cataract is the leading cause of blindness across the world. Age-related cataract (ARC) is the most common type of cataract, but its pathogenesis is not fully understood. Using three-dimensional finite element modeling combining experimental biotechnology, our study demonstrates that external forces during accommodation cause mechanical stress predominantly in lens cortex, basically matching the localization of opacities in cortical ARCs. We identified the cellular senescence and upregulation of PIEZO1 mRNA in HLECs under mechanical stretch. This mechano-induced senescence in HLECs might be mediated by PIEZO1-related pathways, portraying a potential biomechanical cause of cortical ARCs. Our study updates the fundamental insight towards cataractogenesis, paving the way for further exploration of ARCs pathogenesis and nonsurgical treatment.

Dysregulation of Autophagy Occurs During Congenital Cataract Development in ßA3ΔG91 Mice.

Purpose: To examine lens phenotypic characteristics in ßA3ΔG91 mice and determine if ßA3ΔG91 affects autophagy in the lens. Methods: We generated a ßA3ΔG91 mouse model using CRISPR/Cas9 methodology. Comparative phenotypic and biochemical characterizations of lenses from postnatal day 0 (P0), P15, and 1-month-old ßA3ΔG91 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. Results: Relative to WT lenses, 1-month-old ßA3ΔG91 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 ßA3ΔG91 lenses. Comparative IHC and RT-qPCR analyses showed relatively higher levels of autophagy markers (ubiquitinated proteins and p62, LC3, and LAMP2 proteins) in ßA3ΔG91 lenses compared to WT lenses. Additionally, ßA3ΔG91 lenses showed relatively greater numbers of apoptotic cells and higher levels of cleaved caspase-3 and caspase-9. Conclusions: The deletion of G91 in ßA3ΔG91 mice leads to higher levels of expression of autophagy-related proteins and their transcripts relative to WT lenses. Taken together, G91 deletion in ßA3/A1-crystallin is associated with autophagy disruption, attenuation of nuclei degradation, and cellular apoptosis in the lens, which might be congenital cataract causative factors.

The importance of the fourth Greek key motif of human γD-crystallin in maintaining lens transparency-the tale told by the tail.

Purpose: Congenital cataract affects 1-15 per 10,000 newborns worldwide, and 20,000-40,000 children are born every year with developmental bilateral cataracts. Mutations in the crystallin genes are known to cause congenital cataracts. Crystallins, proteins present in the eye lens, are made up of four Greek key motifs separated into two domains. Greek key motifs play an important role in compact folding to provide the necessary refractive index and transparency. The present study was designed to understand the importance of the fourth Greek key motif in maintaining lens transparency by choosing a naturally reported Y134X mutant human γD- crystallin in a Danish infant and its relationship to lens opacification and cataract. Methods: Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods. Results: Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C. Conclusions: Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.

A grape-supplemented diet prevented ultraviolet (UV) radiation-induced cataract by regulating Nrf2 and XIAP pathways.

The purpose of this study is to investigate if grape consumption, in the form of grape powder (GP), could protect against ultraviolet (UV)-induced cataract. Mice were fed with the regular diet, sugar placebo diet, or a grape diet (regular diet supplemented with 5%, 10%, and 15% GP) for 3 months. The mice were then exposed to UV radiation to induce cataract. The results showed that the GP diet dose-dependently inhibited UV-induced cataract and preserved glutathione pools. Interestingly, UV-induced Nrf2 activation was abolished in the groups on the GP diet, suggesting GP consumption may improve redox homeostasis in the lens, making Nrf2 activation unnecessary. For molecular target prediction, a total of 471 proteins regulated by GP were identified using Agilent Literature Search (ALS) software. Among these targets, the X-linked inhibitor of apoptosis (XIAP) was correlated with all of the main active ingredients of GP, including resveratrol, catechin, quercetin, and anthocyanins. Our data confirmed that GP prevented UV-induced suppression of XIAP, indicating that XIAP might be one of the critical molecular targets of GP. In conclusion, this study demonstrated that GP protected the lens from UV-induced cataract development in mice. The protective effects of GP may be attributed to its ability to improve redox homeostasis and activate the XIAP-mediated antiapoptotic pathway.

TGFß overcomes FGF-induced transinhibition of EGFR in lens cells to enable fibrotic secondary cataract.

To cause vision-disrupting fibrotic secondary cataract (PCO), lens epithelial cells that survive cataract surgery must migrate to the posterior of the lens capsule and differentiate into myofibroblasts. During this process, the cells become exposed to the FGF that diffuses out of the vitreous body. In normal development, such relatively high levels of FGF induce lens epithelial cells to differentiate into lens fiber cells. It has been a mystery as to how lens cells could instead undergo a mutually exclusive cell fate, namely epithelial to myofibroblast transition, in the FGF-rich environment of the posterior capsule. We and others have reported that the ability of TGFß to induce lens cell fibrosis requires the activity of endogenous ErbBs. We show here that lens fiber-promoting levels of FGF induce desensitization of ErbB1 (EGFR) that involves its phosphorylation on threonine 669 mediated by both ERK and p38 activity. Transinhibition of ErbB1 by FGF is overcome by a time-dependent increase in ErbB1 levels induced by TGFß, the activation of which is increased after cataract surgery. Our studies provide a rationale for why TGFß upregulates ErbB1 in lens cells and further support the receptor as a therapeutic target for PCO.

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

Increased molar ratio of free fatty acids to albumin in blood as cause and early biomarker for the development of cataracts and Alzheimer's disease.

Cataracts and Alzheimer's disease (AD) are closely linked and are associated with aging and with systemic diseases that increase the molar ratio of free fatty acids to albumin (mFAR) in the blood. From the results of our earlier studies on the development of senile cataracts and from results recently published in the literature on the pathogenesis of Alzheimer's disease, we suggest that there is a common lipotoxic cascade for both diseases, explaining the strong connection between aging, an elevated mFAR in the blood, cataract formation, and AD. Long-chain free fatty acids (FFA) are transported in the blood as FFA/albumin complexes. In young people, vascular albumin barriers in the eyes and brain, very similar in their structure and effect, reduce the FFA/albumin complex concentration from around 650 µmol/l in the blood to 1-3 µmol/l in the aqueous humour of the eyes as well as in the cerebrospinal fluid of the brain. At such low concentrations the fatty acid uptake of the target cells - lens epithelial and brain cells - rises with increasing FFA/albumin complex concentrations, especially when the fatty acid load of albumin molecules is mFAR>1. At higher albumin concentrations, for instance in blood plasma or the interstitial tissue spaces, the fatty acid uptake of the target cells becomes increasingly independent of the FFA/albumin complex concentration and is mainly a function of the mFAR (Richieri et al., 1993). In the blood plasma of young people, the mFAR is normally below 1.0. In people over 40 years old, aging increases the mFAR by decreasing the plasma concentration of albumin and enhancing the plasma concentrations of FFA. The increase in the mFAR in association with C6-unsaturated FFA are risk factors for the vascular albumin barriers (Hennig et al., 1984). Damage to the vascular albumin barrier in the eyes and brain increases the concentration of FFA/albumin complex in the aqueous humour as well as in the cerebrospinal fluid, leading to mitochondrial dysfunction and the death of lens epithelial and brain cells, the development of cataracts, and AD. An age-dependent increase in the concentration of FFA/albumin complex has been found in the aqueous humour of 177 cataract patients, correlating with the mitochondria-mediated apoptotic death of lens epithelial cells, lens opacification and cataracts (Iwig et al., 2004). Mitochondrial dysfunction is also an early crucial event in Alzheimer's pathology, closely connected with the generation of amyloid beta peptides (Leuner et al., 2012). Very recently, amyloid beta production has also been confirmed in the lenses of Alzheimer's patients, causing cataracts (Moncaster et al., 2022). In view of this, we propose that there is a common lipotoxic cascade for senile cataract formation and senile AD, initiated by aging and/or systemic diseases, leading to an mFAR>1 in the blood.

miR-26 Deficiency Causes Alterations in Lens Transcriptome and Results in Adult-Onset Cataract.

Purpose: Despite strong evidence demonstrating that normal lens development requires regulation governed by microRNAs (miRNAs), the functional role of specific miRNAs in mammalian lens development remains largely unexplored. Methods: A comprehensive analysis of miRNA transcripts in the newborn mouse lens, exploring both differential expression between lens epithelial cells and lens fiber cells and overall miRNA abundance, was conducted by miRNA sequencing. Mouse lenses lacking each of three abundantly expressed lens miRNAs (miR-184, miR-26, and miR-1) were analyzed to explore the role of these miRNAs in lens development. Results: Mice lacking all three copies of miR-26 (miR-26TKO) developed postnatal cataracts as early as 4 to 6 weeks of age. RNA sequencing analysis of neonatal lenses from miR-26TKO mice exhibited abnormal reduced expression of a cohort of genes found to be lens enriched and linked to cataract (e.g., Foxe3, Hsf4, Mip, Tdrd7, and numerous crystallin genes) and abnormal elevated expression of genes related to neural development (Lhx3, Neurod4, Shisa7, Elavl3), inflammation (Ccr1, Tnfrsf12a, Csf2ra), the complement pathway, and epithelial to mesenchymal transition (Tnfrsf1a, Ccl7, Stat3, Cntfr). Conclusions: miR-1, miR-184, and miR-26 are each dispensable for normal embryonic lens development. However, loss of miR-26 causes lens transcriptome changes and drives cataract formation.

MicroRNA-1225-5p Promotes the Development of Fibrotic Cataracts via Keap1/Nrf2 Signaling.

PURPOSE: Fibrotic cataracts, including anterior subcapsular cataract (ASC) as well as posterior capsule opacification (PCO), are a common vision-threatening cause worldwide. Still, little is known about the underlying mechanisms. Here, we demonstrate a miRNA-based pathway regulating the pathological fibrosis process of lens epithelium. METHODS: Gain- and loss-of-function approaches, as well as multiple fibrosis models of the lens, were applied to validate the crucial role of two miR-1225 family members in the TGF-ß2 induced PCO model of human LECs and injury-induced ASC model in mice. RESULTS: Both miR-1225-3p and miR-1225-5p prominently stimulate the migration and EMT process of lens epithelial cells (LECs) in vitro as well as lens fibrosis in vivo. Moreover, we demonstrated that the underlying mechanism for these effects of miR-1225-5p is via directly targeting Keap1 to regulate Keap1/Nrf2 signaling. In addition, evidence showed that Keap1/Nrf2 signaling is activated in the TGF-ß2 induced PCO model of human LECs and injury-induced ASC model in mice, and inhibition of the Nrf2 pathway can significantly reverse the process of LECs EMT as well as lens fibrosis. CONCLUSIONS: These results suggest that blockade of miR-1225-5p prevents lens fibrosis via targeting Keap1 thereby inhibiting Nrf2 activation. The 'miR-1225-Keap1-Nrf2' signaling axis presumably holds therapeutic promise in the treatment of fibrotic cataracts.

CircSTRBP contributes to H2O2-induced lens epithelium cell dysfunction through increasing NOX4 mRNA stability by recruiting IGF2BP1.

Previous studies have shown that the development of age-related cataract (ARC) is involved in lens epithelium dysfunction, which is associated with abnormally expressed circular RNAs (circRNAs). The current work aims to probe the role of circSTRBP (hsa_circ_0088,427) in hydrogen peroxide (H2O2)-induced lens epitheliums. Lens epithelium tissues were harvested from ARC or normal subjects (n = 23). CircSTRBP, spermatid perinuclear RNA binding protein (STRBP), and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation, cycle progression, and apoptosis were assessed using 5-ethynyl-2'-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and flow cytometry assays. Caspase 3 activity, reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidases (GSH-PX) levels were detected using corresponding kits. NOX4 protein level was determined using Western blot. The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and circSTRBP or NOX4 was assessed through RNA immunoprecipitation (RIP). CircSTRBP and NOX4 abundances were increased in lens epithelium samples from ARC patients and H2O2-treated SRA01/04 cells. CircSTRBP knockdown might abolish H2O2-triggered SRA01/04 cell proliferation repression and apoptosis and oxidative stress promotion. In mechanism, circSTRBP is bound with IGF2BP1 and improves the stability and expression of NOX4 mRNA in SRA01/04 cells. CircSTRBP facilitated H2O2-induced SRA01/04 cell apoptosis and oxidative stress through by enhancing NOX4 mRNA stability via recruiting IGF2BP1, providing novel insights for ARC progression and treatment.

In vivo quasi-elastic light scattering detects molecular changes in the lenses of adolescents with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder in humans. DS is associated with increased prevalence of several ocular sequelae, including characteristic blue-dot cerulean cataract. DS is accompanied by age-dependent accumulation of Alzheimer's disease (AD) amyloid-ß (Aß) peptides and amyloid pathology in the brain and comorbid early-onset Aß amyloidopathy and colocalizing cataracts in the lens. Quasi-elastic light scattering (QLS) is an established optical technique that noninvasively measures changes in protein size distributions in the human lens in vivo. In this cross-sectional study, lenticular QLS correlation time was decreased in adolescent subjects with DS compared to age-matched control subjects. Clinical QLS was consistent with alterations in relative particle hydrodynamic radius in lenses of adolescents with DS. These correlative results suggest that noninvasive QLS can be used to evaluate molecular changes in the lenses of individuals with DS.

Conditional Ablation of Spred1 and Spred2 in the Eye Lens Negatively Impacts Its Development and Growth.

The development and growth of the eye depends on normal lens morphogenesis and its growth. This growth, in turn, is dependent on coordinated proliferation of the lens epithelial cells and their subsequent differentiation into fiber cells. These cellular processes are tightly regulated to maintain the precise cellular structure and size of the lens, critical for its transparency and refractive properties. Growth factor-mediated MAPK signaling driven by ERK1/2 has been reported as essential for regulating cellular processes of the lens, with ERK1/2 signaling tightly regulated by endogenous antagonists, including members of the Sprouty and related Spred families. Our previous studies have demonstrated the importance of both these inhibitory molecules in lens and eye development. In this study, we build on these findings to highlight the importance of Spreds in regulating early lens morphogenesis by modulating ERK1/2-mediated lens epithelial cell proliferation and fiber differentiation. Conditional loss of both Spred1 and Spred2 in early lens morphogenesis results in elevated ERK1/2 phosphorylation, hyperproliferation of lens epithelia, and an associated increase in the rate of fiber differentiation. This results in transient microphakia and microphthalmia, which disappears, owing potentially to compensatory Sprouty expression. Our data support an important temporal role for Spreds in the early stages of lens morphogenesis and highlight how negative regulation of ERK1/2 signaling is critical for maintaining lens proliferation and fiber differentiation in situ throughout life.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens.

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Glutamate is effective in decreasing opacity formed in galactose-induced cataract model.

Although cataract is the leading cause of blindness worldwide, the detailed pathogenesis of cataract remains unclear, and clinically useful drug treatments are still lacking. In this study, we examined the effects of glutamate using an ex vivo model in which rat lens is cultured in a galactose-containing medium to induce opacity formation. After inducing lens opacity formation in galactose medium, glutamate was added, and the opacity decreased when the culture was continued. Next, microarray analysis was performed using samples in which the opacity was reduced by glutamate, and genes whose expression increased with galactose culture and decreased with the addition of glutamate were extracted. Subsequently, STRING analysis was performed on a group of genes that showed variation as a result of quantitative measurement of gene expression by RT-qPCR. The results suggest that apoptosis, oxidative stress, endoplasmic reticulum (ER) stress, cell proliferation, epithelial-mesenchymal transition (EMT), cytoskeleton, and histones are involved in the formation and reduction of opacity. Therefore, glutamate may reduce opacity by inhibiting oxidative stress and its downstream functions, and by regulating the cytoskeleton and cell proliferation.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.