EphA2 Affects Development of the Eye Lens Nucleus and the Gradient of Refractive Index.

Purpose: Our studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index. Methods: We performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Morphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2-/- lenses and ephrin-A5-/- lenses. Surprisingly, EphA2-/- lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2-/- fiber cells close to the center of the lens. Inner EphA2-/- lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5-/- lenses. There was an overall decrease in magnitude of refractive index across EphA2-/- lenses, which is most pronounced in the nucleus. Conclusions: This work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.

Acceleration of age-induced proteolysis in the guinea pig lens nucleus by in vivo exposure to hyperbaric oxygen: A mass spectrometry analysis.

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.

Differences in the properties of porcine cortical and nuclear fiber cell plasma membranes revealed by saturation recovery EPR spin labeling measurements.

Eye lens membranes are complex biological samples. They consist of a variety of lipids that form the lipid bilayer matrix, integral proteins embedded into the lipid bilayer, and peripheral proteins. This molecular diversity in membrane composition induces formation of lipid domains with particular physical properties that are responsible for the maintenance of proper membrane functions. These domains can be, and have been, effectively described in terms of the rotational diffusion of lipid spin labels and oxygen collision with spin labels using the saturation recovery (SR) electron paramagnetic resonance method and, now, using stretched exponential function for the analysis of SR signals. Here, we report the application of the stretched exponential function analysis of SR electron paramagnetic resonance signals coming from cholesterol analog, androstane spin label (ASL) in the lipid bilayer portion of intact fiber cell plasma membranes (IMs) isolated from the cortex and nucleus of porcine eye lenses. Further, we compare the properties of these IMs with model lens lipid membranes (LLMs) derived from the total lipids extracted from cortical and nuclear IMs. With this approach, the IM can be characterized by the continuous probability density distribution of the spin-lattice relaxation rates associated with the rotational diffusion of a spin label, and by the distribution of the oxygen transport parameter within the IM (i.e., the collision rate of molecular oxygen with the spin label). We found that the cortical and nuclear LLMs possess very different, albeit homogenous, spin lattice relaxation rates due to the rotational diffusion of ASL, indicating that the local rigidity around the spin label in nuclear LLMs is considerably greater than that in cortical LLMs. However, the oxygen transport parameter around the spin label is very similar and slightly heterogenous for LLMs from both sources. This heterogeneity was previously missed when distinct exponential analysis was used. The spin lattice relaxation rates due to either the rotational diffusion of ASL or the oxygen collision with the spin label in nuclear IMs have slower values and wider distributions compared with those of cortical IMs. From this evidence, we conclude that lipids in nuclear IMs are less fluid and more heterogeneous than those in cortical membranes. Additionally, a comparison of properties of IMs with corresponding LLMs, and lipid and protein composition analysis, allow us to conclude that the decreased lipid-to-protein ratio not only induces greater rigidity of nuclear IMs, but also creates domains with the considerably decreased and variable oxygen accessibility. The advantages and disadvantages of this method, as well as its use for the cluster analysis, are discussed.

Constant lens fiber cell thickness in fish suggests crystallin transport to denucleated cells.

The crystalline lens of the vertebrate eye grows throughout life. This growth may be enormous in fish, while the lens must be functional from larva to adult. During growth, the fiber cells of the lens must increase the concentration of specific proteins (crystallins) in the cytoplasm to increase refractive index. However, the bulk of the fiber cells in a vertebrate lens are denucleated and have no organelles to synthesize proteins. To study how this problem is solved, we first measured lens fiber cell thickness in the Nile tilapia, a teleost fish. In the lenses from 25 fish, in two size groups, fibers were considerably thinner than in other vertebrates. Fiber thickness was about constant along the radius of the lens and the same between the size groups. Since our results provided no evidence for shrinkage of lens fiber cells with growth (expected if protein concentration is increased by expelling water) we included eight additional teleost species to elucidate the mechanism by which the cells increase crystallin concentration. In all species, fiber cell thickness was about constant throughout the lens, with species-specific values. The changes in fiber cell thickness expected from an increase in crystallin concentration by removal of water were modeled. Shrinkage in cell thickness by up to 66% would have been necessary to reach the required crystallin concentration. We conclude that crystallin concentration in denucleated lens fiber cells is increased by transport of proteins from synthetically competent cells in the periphery of the lens.

Age-related spatial differences of human lens UV filters revealed by negative ion mode MALDI imaging mass spectrometry.

Tryptophan-derived UV filters are predominantly found in the lenses of primates and humans. While protective against UV radiation, aging alters the complement and spatial distributions of human lens UV filters, and a role for UV filters has been suggested in age-related cataract formation. To establish how the spatial distributions of UV filters change in normal human lens aging, matrix assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) was utilised to map the locations and relative abundance of multiple UV filters simultaneously. Frozen human lenses were cryosectioned axially, and the 20 µm-thick sections coated with MALDI matrix via robotic sprayer and analysed using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS. While signal for many UV filters was detected throughout the lenses, signal intensity was generally highest in the central (embryonic) nucleus and decreased uniformly in outer (foetal, juvenile, adult) nuclear and cortical regions, and many UV filter signals declined with age. In contrast, two antioxidant-conjugated UV filters (Cys-3-OHKG and GSH-3-OHKG) were restricted to the lens nucleus and their relative signal increased with increasing lens age. The enhanced spatial resolution of MALDI-IMS over manual trephine dissection techniques and its multiplex capability allowed the spatial relationships between lens UV filters to be established and explored in relation to aging. Together these results confirmed that the complement of UV filters in each lens is dynamic and undergoes significant age-related changes. In the future, this information could be used to compare with other lens biomolecule changes to better understand the lens aging process and age-related cataract formation.

Quantitative proteomics analysis with iTRAQ in human lenses with nuclear cataracts of different axial lengths.

PURPOSE: The goal of this study was to identify and quantify the differentially expressed proteins in human nuclear cataract with different axial lengths. METHODS: Thirty-six samples of human lens nuclei with hardness grade III or IV were obtained during cataract surgery with extracapsular cataract extraction (ECCE). Six healthy transparent human lens nuclei were obtained from fresh healthy cadaver eyes during corneal transplantation surgery. The lens nuclei were divided into seven groups (six lenses in each group) according to the optic axis: Group A (mean axial length 28.7±1.5 mm; average age 59.8±1.9 years), Group B (mean axial length 23.0±0.4 mm; average age 60.3±2.5 years), Group C (mean axial length 19.9±0.5 mm; average age 55.1±2.5 years), Group D (mean axial length 28.7±1.4 mm; average age 58.0±4.0 years), Group E (mean axial length 23.0±0.3 mm; average age 56.9±4.2 years), and Group F (mean axial length 20.7±0.6 mm; average age 57.6±5.3 years). The six healthy transparent human lenses were included in a younger group with standard optic axes, Group G (mean axial length 23.0±0.5 mm; average age 34.7±4.2 years).Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from the samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were analyzed using 8-plex isobaric tagging for relative and absolute protein quantification (iTRAQ) labeling combined with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS). The data were analyzed with ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated with western blotting. RESULTS: We employed biological and technical replicates and selected the intersection of the two sets of results, which included 40 proteins. From the 40 proteins identified, six were selected as differentially expressed proteins closely related to axial length. The six proteins were gap junction alpha-3 protein, beta-crystallin B2, T-complex protein 1 subunit beta, gamma-enolase, pyruvate kinase isozymes M1/M2, and sorbitol dehydrogenase. Levels of beta-crystallin B2 expression were decreased in nuclear cataracts with longer axial length. The results of the mass spectrometric analysis were consistent with the western blot validation. CONCLUSION: The discovery of these differentially expressed proteins provides valuable clues for understanding the pathogenesis of axial-related nuclear cataract. The results indicate that beta-crystallin B2 (CRBB2) may be involved in axial-related nuclear cataract pathogenesis. Further studies are needed to investigate the correlation between CRBB2 and axial-related nuclear cataract.

Longitudinal Study of Age-Related Cataract Using Dynamic Light Scattering: Loss of α-Crystallin Leads to Nuclear Cataract Development.

PURPOSE: To conduct a longitudinal study on age-related nuclear cataracts using dynamic light scattering (DLS) to determine if cataract progression is associated with loss of the unbound form of the lens molecular chaperone protein, α-crystallin. DESIGN: Natural history and cohort study. PARTICIPANTS: Patients 30 years of age or older of either gender seeking treatment at the Wilmer Eye Institute Cornea-Cataract Department. METHODS: All patients underwent a comprehensive dilated eye examination every 6 months, including slit-lamp grading of their lenses using the Age-Related Eye Disease Study (AREDS) clinical lens grading system and obtaining an estimate of unbound α-crystallin level in the nucleus, the α-crystallin index (ACI), using the National Aeronautics and Space Administration-National Eye Institute DLS device. We used a random effects statistical model to examine the relationship of lens opacity changes over time with ACI changes. MAIN OUTCOME MEASURES: α-Crystallin Index (ACI) and AREDS nuclear cataract grade. RESULTS: Forty-five patients (66 eyes) 34 to 79 years of age with AREDS nuclear lens grades of 0 to 3.0 were followed up every 6 months for a mean of 19 months (range, 6-36 months). We found that lenses with the lowest baseline levels of ACI had the most rapid progression of cataracts, whereas lenses with higher ACI at baseline had no or slower cataract progression. Lenses that lost α-crystallin at the highest rates during the study also had faster progression of nuclear cataracts than lenses with a slower rate of ACI loss. Kaplan-Meier survival curves showed that lenses with the lowest initial ACI had the highest risk of undergoing cataract surgery. CONCLUSIONS: This longitudinal study corroborates our previous cross-sectional study finding that higher levels of unbound α-crystallin as assessed by ACI are associated with lower risk of cataract formation and that loss of ACI over time is associated with cataract formation and progression. This study suggested that assessment of ACI with the DLS device could be used as a surrogate for lens opacity risk in clinical studies, and for assessing nuclear cataract events in studies where cataract development may be a side effect of a drug or device.

Protein profiles in cortical and nuclear regions of aged human donor lenses: A confocal Raman microspectroscopic and imaging study.

A combination of Raman spectroscopy, imaging, hierarchical cluster analysis (HCA) and peak ratio analysis was used to analyze protein profiles in the superficial cortex (SC), deep cortex (DC) and nucleus of old human lenses with cortical, nuclear and mixed cataracts. No consistent differences were observed in protein spectra and after cluster analysis between the three locations irrespective of the presence or absence of cortical opacities and/or coloration. A sharp increase (∼15%-∼33%) in protein content from SC to DC, normal for human lenses, was found in 7 lenses. In 4 lenses, characterized by the absence of cortical opacities, the SC has a protein content of ∼35%. A significant increase in the disulfide-to-protein ratio is found only in the SC of the 7 cortical cataracts. No changes were found in sulfhydryl-to-protein ratio. The relative contents of α-helices and ß-sheets increase from SC to nucleus. ß-Sheets are more common in the SC of lenses with cortical cataract. The absence of significant and consistent changes in protein profiles between nucleus and cortex even in cases of severe coloration is not favoring the prevailing concept that ubiquitous protein oxidation is a key factor for age related nuclear (ARN) cataracts. The observations favor the idea that multilamellar bodies or protein aggregates at very low volume densities are responsible for the rise in Mie light scatter as a main cause of ARN cataracts leaving the short-range-order of the fiber cytoplasm largely intact. The absence of significant changes in the protein spectra of the deep cortical opacities, milky white as a result of the presence of vesicle-like features, indicate they are packed with relatively undisturbed crystallins.

Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: Methodology development and its application to studies of porcine lens membranes.

An electron paramagnetic resonance spin-labeling method has been developed that allows quantitative evaluation of the amounts of phospholipids and cholesterol in lipid domains of intact fiber-cell plasma membranes isolated from cortical and nuclear regions of eye lenses. The long term goal of this research is the assessment of organizational changes in human lens fiber cell membranes that occur with age and during cataract development. The measurements needed to be performed on lens membranes prepared from eyes of single donors and from single eyes. For these types of studies it is necessary to separate the age/cataract related changes from preparation/technique related changes. Human lenses differ not only because of age, but also because of the varying health histories of the donors. To solve these problems the sample-to-sample preparation/technique related changes were evaluated for cortical and nuclear lens membranes prepared from single porcine eyes. It was assumed that the differences due to the age (animals were two year old) and environmental conditions for raising these animals were minimal. Mean values and standard deviations from preparation/technique changes for measured amounts of lipids in membrane domains were calculated. Statistical analysis (Student's t-test) of the data also allowed determining the differences of mean values which were statistically significant with P ≤ 0.05. These differences defined for porcine lenses will be used for comparison of amounts of lipids in domains in human lens membranes prepared from eyes of single donors and from single eyes. Greater separations will indicate that differences were statistically significant with (P ≤ 0.05) and that they came from different than preparation/technique sources. Results confirmed that in nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human intact fiber cell membranes [Raguz, M. Mainali, L., O'Brien, W.J., and Subczynski, W.K. (2015) Exp. Eye Res.]. Lipids in porcine nuclear fiber cell plasma membranes were more rigid and less permeable to oxygen than in human nuclear membranes. Most likely the significant differences in the lipid composition were responsible for the observed differences.

Lipid domains in intact fiber-cell plasma membranes isolated from cortical and nuclear regions of human eye lenses of donors from different age groups.

The results reported here clearly document changes in the properties and the organization of fiber-cell membrane lipids that occur with age, based on electron paramagnetic resonance (EPR) analysis of lens membranes of clear lenses from donors of age groups from 0 to 20, 21 to 40, and 61 to 80 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provide an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of lipids in these domains. Fiber-cell membranes were found to contain three distinct lipid environments: bulk lipid domain, which appears minimally affected by membrane proteins, and two domains that appear due to the presence of membrane proteins, namely boundary and trapped lipid domains. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains increased with the donors' age and was greater than that in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins in nuclear and cortical membranes increased with the donors' age. It was also shown that cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. It is evident that the rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The amount of lipids in domains of low oxygen permeability, mainly in trapped lipid domains, were greater in nuclear than cortical membranes and increased with the age of donors. These results indicate that the nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and become less permeable to oxygen with age. In clear lenses, age-related changes in the lens lipid and protein composition and organization appear to occur in ways that increase fiber cell plasma membrane resistance to oxygen permeation.

Spatial distributions of AQP5 and AQP0 in embryonic and postnatal mouse lens development.

The expression of the water channel protein aquaporin (AQP)-5 in adult rodent and human lenses was recently reported using immunohistochemistry, molecular biology, and mass spectrometry techniques, confirming a second transmembrane water channel that is present in lens fibre cells in addition to the abundant AQP0 protein. Interestingly, the sub-cellular distribution and level of post-translational modification of both proteins changes with fibre cell differentiation and location in the adult rodent lens. This study compares the sub-cellular distribution of AQP0 and AQP5 during embryonic and postnatal fibre cell development in the mouse lens to understand how the immunolabelling patterns for both AQPs observed in adult lens are first established. Immunohistochemistry was used to map the cellular and sub-cellular distribution of AQP5 and AQP0 throughout the lens in cryosections from adult (6 weeks-8 months) and postnatal (0-2 weeks) mouse lenses and in sections from paraffin embedded mouse embryos (E10-E19). All sections were imaged by fluorescence confocal microscopy. Using antibodies directed against the C-terminus of each AQP, AQP5 was abundantly expressed early in development, being found in the cytoplasm of cells of the lens vesicle and surrounding tissues (E10), while AQP0 was detected later (E11), and only in the membranes of elongating primary fibre cells. During the course of subsequent embryonic and postnatal development the pattern of cytoplasmic AQP5 and membranous AQP0 labelling was maintained until postnatal day 6 (P6). From P6 AQP5 labelling became progressively more membranous initially in the lens nucleus and then later in all regions of the lens, while AQP0 labelling was abruptly lost in the lens nucleus due to C-terminal truncation. Our results show that the spatial distribution patterns of AQP0 and AQP5 observed in the adult lens are established during a narrow window of postnatal development (P6-P15) that precedes eye opening and coincides with regression of the hyaloid vascular system. Our results support the hypothesis that, in the older fibre cells, insertion of AQP5 into the fibre cell membrane may compensate for any change in the functionality of AQP0 induced by truncation of its C-terminal tail.

Quantitative proteomics analysis by iTRAQ in human nuclear cataracts of different ages and normal lens nuclei.

PURPOSE: The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans. EXPERIMENTAL DESIGN: Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting. RESULTS: We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation. CONCLUSION AND CLINICAL RELEVANCE: The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

Shotgun proteomic analysis of S-thiolation sites of guinea pig lens nuclear crystallins following oxidative stress in vivo.

PURPOSE: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO). METHODS: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls. RESULTS: All major crystallins, except αB, were modified to some extent by either S-glutathiolation or S-cysteinylation. Overall, 72% of the cysteine residues of guinea pig lens nuclear crystallins were shown to be capable of binding glutathione, cysteine, or both molecules. The crystallin with the highest level of modification was ßA1/A3 (six of eight -SH groups), and that with the lowest (two of five -SH groups) was ßA2. O(2)-induced increases in PSSG levels were 2.8, 2.4, and 4.1 times control for γA-, γB-, and γC-crystallins, respectively. Comparable increases in PSSC levels for the three γ-crystallins were 2.3, 2.7, and 2.4 times control, respectively. ßB2-crystallin showed the highest amount of O(2)-induced PSSG formation of any of the crystallins, as well as a substantial level of control PSSG, and nearly all of this was due to a single residue, C67, a site also present in human ßB2-crystallin. Overall, 32 of the 44 modified cysteine residues were homologous with the human. CONCLUSIONS: This large-scale study successfully identified lens crystallin cysteine residues that bound glutathione and/or cysteine under normal or oxidative stress conditions. The high percentage of protein -SH groups that are modified by S-thiolation in the guinea pig lens nucleus demonstrates the substantial protein sulfhydryl redox buffer capability present in the center of the lens. The results suggest that PSSG and PSSC formation may act to delay O(2)-induced insolubilization of γA-, γB-, and γC-crystallins, and ß-crystallins, but with a greater effect on the γ-crystallins at an early stage of oxidative stress. The study has shown that technological approaches are now available to investigate in considerable detail the role of specific lens -SH groups in nuclear cataractogenesis.

Carbon turnover in the water-soluble protein of the adult human lens.

PURPOSE: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse. METHODS: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse. RESULTS: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. CONCLUSIONS: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.

Properties of fiber cell plasma membranes isolated from the cortex and nucleus of the porcine eye lens.

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eye lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes, because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes-namely, the domain formed by boundary lipids and the domain formed by trapped lipids-were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.

Instability of the cellular lipidome with age.

The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.

Proteomic analysis of human age-related nuclear cataracts and normal lens nuclei.

PURPOSE: To identify proteomic differences between age-related nuclear cataracts (ARNCs) and normal lens nuclei. METHODS: Total solubilized proteins from ARNC lens nuclei with different grades were compared with normal controls by 2-D differential in-gel electrophoresis (2-D DIGE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses determined the compositions of high molecular weight (HMW; >200 kDa) aggregates found in ARNC lens nuclei. Western blot analysis was used to verify the changes in αA-crystallin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. RESULTS: The 2-D differential in-gel electrophoresis results showed that nine proteins were significantly less abundant in lens nuclei from ARNC patients than in control lens nuclei. Six proteins (αA-, ßA3-, ßA4-, ßB1-, and γD-crystallin and putative uncharacterized protein DKFZp434A0627 from the CRYGS family) tended to decrease as the cataract grade increased, while the other three proteins (αB-crystallin, GAPDH, and retinal dehydrogenase 1) did not show such a tendency. SDS-PAGE showed decreased protein levels at ∼20 kDa in ARNC lenses but significantly increased levels at HMW (>200 kDa). Liquid chromatography tandem mass spectrometry analysis showed that the HMW aggregates derived largely from crystallins also contained filensin, phakinin, and carbonyl reductase 1. Of all the components, αA-crystallin accounted for the highest fraction. αA-, αB-, and γD-crystallin and DKFZp434A0627 were more prone to aggregate than other crystallins. CONCLUSIONS: The results show that crystallins, especially αA-crystallin, aggregate irreversibly during ARNC development. Some enzymes (GAPDH, retinal dehydrogenase 1, and carbonyl reductase 1) may be involved in and/or accelerate this process.

Understanding the α-crystallin cell membrane conjunction.

PURPOSE: It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS: Fiber cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea, and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS: The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an NH2-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS: Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure.

X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.

PURPOSE: To compare age-related cataractous (ARC) changes in unirradiated mice lenses to those induced by head-only X-irradiation of 3 month-old mice. METHODS: lens epithelial cells (LECs) as well as partially degraded cortical DNA were visualized in fixed sections using 4',6-diamidino-2-phenylindole (DAPI) staining, and in fresh lenses using the vital stain Hoechst 33342. reactive oxygen species (ROS) activity was also visualized directly in fresh lenses using the vital dye Dihydrorhodamine (DHR). In fixed lenses an antibody specific for 8-OH Guanosine (8-OH-G) lesions was used to visualize DNA oxidative adducts from ROS damage. Alpha smooth muscle actin was visualized using specific antibodies to determine if myofibroblasts were present. Fluorescence was quantified using Laser Scanning Confocal Microscopy (LSCM). The degree of lens opacity and cataract formation was determined by slit lamp, or from digitalized images of light reflections taken with a low magnification light microscope. RESULTS: Using DNA- and ROS-specific vital fluorescent dyes, and laser scanning confocal microscopy we have previously described 4 changes in the aging rodent lenses: 1) a significantly decreased density of surface LECs in lenses from old compared to younger mice and rats; 2) a very large increase in retained cortical nuclei and DNA fragments in the secondary lens fibers of old rodent lenses; 3) increased cortical ROS in old rodent lenses; 4) increased cataract concomitantly with the cortical DNA and ROS increases. In the current study we report that these same 4 changes also occur in an accelerated fashion in mice given head-only X-irradiation at 3 months of age. In addition to vital staining of fresh lenses, we also examined sections from fixed eyes stained with DAPI or hematoxylin and eosin (H&E) and found the same loss of surface LECs and accumulation of undigested nuclei and debris in secondary lens fibers occur with age or following X-irradiation. In addition sections from fixed-eyes were examined for ROS damage to DNA with antibodies specific for 8-OH-G lesions. The frequency of 8-OH-G lesions increased dramatically in lenses from old unirradiated mice over 24 months of age, and similarly in X-irradiated lenses by 9-11 months post irradiation. The accumulation of cortical nuclei was not the result of conversion or invasion by myofibroblasts as tested by antibodies to a marker for such cells, alpha smooth muscle actin. CONCLUSIONS: X-irradiation damage induces a large decrease in surface LECs over a period of 3-11 months post X-irradiation of young mice. These changes are similar in extent to those seen in 24-29 months-old control mouse lenses with age-related cataracts. In 24+ month-old unirradiated mice the secondary lens fibers are not able to degrade nuclei or nuclear DNA efficiently and accumulate large numbers of cortical nuclei and nuclear fragments as well as ROS and 8-OHG lesions. X-irradiated lenses develop the same abnormalities in a more accelerated fashion. The extensive loss of LECS and accumulation of undegraded nuclei, ROS, and ROS damage may play a causal role in cataract generation in both unirradiated old mice and in previously irradiated young adult mice.

Effect of age on the thioltransferase (glutaredoxin) and thioredoxin systems in the human lens.

PURPOSE: To investigate the effect of age on the key oxidation repair enzymes of the thioltransferase (TTase) and thioredoxin (TRx) systems in the human lens. METHODS: Twenty-three normal human lenses (donor ages, 19-77 years) were grouped into second, third, fifth, sixth, and seventh decades and analyzed for TTase, TRx, glutathione reductase (GR), thioredoxin reductase (TR), and glyceraldehyde-3-phosphate dehydrogenase (G3PD) activities, as well as the glutathione (GSH) pool. Additionally, 19 contralateral lenses of the donor eyes were each divided into cortex and nucleus for enzyme distribution studies. RESULTS: All the enzymes showed similar activity in the cortex and nucleus, regardless of age, but were inactivated to various extents in the older lenses. In the TTase system, both TTase and GR showed activity loss over the five decades, with 70% remaining in the seventh decade, whereas the GSH pool was depleted extensively, with only 35% left in the older lenses. In the TRx system, TRx activity was not affected as much as TR for which only 70% of the activity was found in the seventh decade compared with the second to third decades. Overall, G3PD was more sensitive to age because only 50% activity remained after the sixth decade. CONCLUSIONS: With increasing age there is a gradual activity loss in both the TTase and the TRx systems and a lowered GSH pool. These alterations, compounded with the age-related loss in G3PD activity, may lead to redox and energy imbalance, likely contributing to a higher risk to cataract formation in the aging population.