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.

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.

Effects of lentiviral short hairpin RNA silencing of Toll-like receptor 4 on the lens epithelial cell line HLEC.

The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.

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.

Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing.

PURPOSE: The ocular lens contains only two cell types: epithelial cells and fiber cells. The epithelial cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens fiber cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial cells and lens fiber cells was conducted to comprehensively compare the transcriptomes of these two cell types. METHODS: RNA from three biologic replicate samples of epithelial and fiber cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR). RESULTS: Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial cells into fiber cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial cells and lens fiber cells. To our knowledge, this is the first study identifying the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes upregulated in the epithelial cells were enriched for extracellular matrix production, cell division, migration, protein kinase activity, growth factor binding, and calcium ion binding. Genes upregulated in the fiber cells were enriched for proteosome complexes, unfolded protein responses, phosphatase activity, and ubiquitin binding. Differentially expressed genes involved in several important signaling pathways, lens structural components, organelle loss, and denucleation were also highlighted to provide insights into lens development and lens fiber differentiation. CONCLUSIONS: RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of protein-coding and non-coding transcripts from lens epithelial cells and lens fiber cells. This information provides a valuable resource for studying lens development, nuclear degradation, and organelle loss during fiber differentiation, and associated diseases.

Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse.

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1-16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 µm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

Absence of beta-amyloid in cortical cataracts of donors with and without Alzheimer's disease.

Eye lenses from human donors with and without Alzheimer's disease (AD) were studied to evaluate the presence of amyloid in cortical cataract. We obtained 39 lenses from 21 postmortem donors with AD and 15 lenses from age-matched controls provided by the Banco de Ojos para Tratamientos de la Ceguera (Barcelona, Spain). For 17 donors, AD was clinically diagnosed by general physicians and for 4 donors the AD diagnosis was neuropathologically confirmed. Of the 21 donors with AD, 6 had pronounced bilateral cortical lens opacities and 15 only minor or no cortical opacities. As controls, 7 donors with pronounced cortical opacities and 8 donors with almost transparent lenses were selected. All lenses were photographed in a dark field stereomicroscope. Histological sections were analyzed using a standard and a more sensitive Congo red protocol, thioflavin staining and beta-amyloid immunohistochemistry. Brain tissue from two donors, one with cerebral amyloid angiopathy and another with advanced AD-related changes and one cornea with lattice dystrophy were used as positive controls for the staining techniques. Thioflavin, standard and modified Congo red staining were positive in the control brain tissues and in the dystrophic cornea. Beta-amyloid immunohistochemistry was positive in the brain tissues but not in the cornea sample. Lenses from control and AD donors were, without exception, negative after Congo red, thioflavin, and beta-amyloid immunohistochemical staining. The results of the positive control tissues correspond well with known observations in AD, amyloid angiopathy and corneas with lattice dystrophy. The absence of staining in AD and control lenses with the techniques employed lead us to conclude that there is no beta-amyloid in lenses from donors with AD or in control cortical cataracts. The inconsistency with previous studies of Goldstein et al. (2003) and Moncaster et al. (2010), both of which demonstrated positive Congo red, thioflavin, and beta-amyloid immunohistochemical staining in AD and Down syndrome lenses, is discussed.

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.

Lens Aquaporin-5 Inserts Into Bovine Fiber Cell Plasma Membranes Via Unconventional Protein Secretion.

Purpose: To spatially map aquaporin-5 (AQP5) expression in the bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms. Methods: Immunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, Sec22ß, LIMP-2, and connexin 50 antibodies and the membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens sections to examine cytoplasmic organelle morphology and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10-nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control for 24 hours in ex vivo culture to determine changes in AQP5 plasma membrane expression. Results: Immunofluorescence analysis revealed cytoplasmic AQP5 expression in lens epithelial cells and differentiating fiber cells. In the lens cortex, complete AQP5 plasma membrane insertion occurs at r/a = 0.951 ± 0.005. AQP5-containing cytoplasmic vesicles are spheroidal in morphology with linear extensions, express TOMM20, and contain LC3B and LIMP-2, but not Sec22ß, as fiber cells mature. TEM analysis revealed complex vesicular assemblies with congruent subcellular localization to AQP5-containing cytoplasmic vesicles. AQP5-containing cytoplasmic vesicles appear to dock with the plasma membrane. Bafilomycin A1 treatment reduced AQP5 plasma membrane expression by 27%. Conclusions: AQP5 localizes to spheroidal, linear cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and presumably fuse with LIMP-2-positive lysosomes. Our data suggest that AQP5 to the plasma membrane through lysosome-associated unconventional protein secretion, a novel mechanism of AQP5 trafficking.

Altered chaperone-like activity of alpha-crystallins promotes cataractogenesis.

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.

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.

Age-related retention of fiber cell nuclei and nuclear fragments in the lens cortices of multiple species.

PURPOSE: To determine the differences between species in the retention of lens fiber cell nuclei and nuclear fragments in the aging lens cortex and the relationship of nuclear retention to lens opacity. For this purpose old human, monkey, dog, and rat lenses were compared to those of three strains of mouse. We also investigated possible mechanisms leading to nuclear retention. METHODS: Fixed specimens of the species referred to above were obtained from immediate on site sacrifice of mice and rats, or from recently fixed lenses of other species, dogs, monkeys, and humans, obtained from collaborators. The retention of undegraded nuclei and nuclear fragments was graded 1-4 from histologic observation. All species lenses were examined microscopically in fixed sections stained with hematoxylin and eosin (H&E) or 4',6-diamidino-2-phenylindole (DAPI). Slit lamp observations were made only on the mice and rats before sacrifice and lens fixation. Values of 0 to 4 (clear lens to cataract) were given to degree of opacity. MRNA content in young versus old C57BL/6 mouse lenses was determined by quantitative PCR (qPCR) for DNase II-like acid DNase (DLAD) and other proteins. DLAD protein was determined by immunofluorescence of fixed eye sections. RESULTS: In old C57BL/6 and DBA mice and, to a lesser degree, in old CBA mice and old Brown Norway (BN) rats lenses were seen to contain a greatly expanded pool of unresolved whole nuclei or fragments of nuclei in differentiating lens fiber cells. This generally correlated with increased slit lamp opacities in these mice. Most old dog lenses also had an increase in retained cortical nuclei, as did a few old humans. However, a second rat strain, BNF1, in which opacity was quite high had no increase in retained nuclei with age nor did any of the old monkeys, indicating that retained nuclei could not be a cause of opacity in these animals. The nuclei and nuclear fragments were located at all levels in the outer cortex extending inward from the lens equator and were observable by the DAPI. These nuclei and nuclear fragments were seen from 12 months onward in all C57BL/6 and DBA/2 mice and to a lesser degree in the CBA, increasing in number and in space occupancy with increasing age. Preliminary results suggest that retention of nuclei in the C57BL/6 mouse is correlated with an age-related loss of DLAD from old lenses. CONCLUSIONS: A very marked apparently light refractive condition caused by retained cortical nuclei and nuclear fragments is present in the lens cortices, increasing with age in the three strains of mice examined and in one of two strains of rats (BN). This condition was also seen in some old dogs and a few old humans. It may be caused by an age-related loss of DLAD, which is essential for nuclear DNA degradation in the lens. However, this condition does not develop in old BNF1 rats, or old monkeys and is only seen sporadically in humans. Thus, it can not be a universal cause for age related lens opacity or cataract presence, although it develops concurrently with opacity in mice. This phenomenon should be considered when using the old mouse as a model for human age-related cataract.

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.

Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.

PURPOSE: To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. RESULTS: Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. CONCLUSIONS: We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.

On the growth and internal structure of the human lens.

Growth of the human lens and the development of its internal features are examined using in vivo and in vitro observations on dimensions, weights, cell sizes, protein gradients and other properties. In vitro studies have shown that human lens growth is biphasic, asymptotic until just after birth and linear for most of postnatal life. This generates two distinct compartments, the prenatal and the postnatal. The prenatal growth mode leads to the formation of an adult nuclear core of fixed dimensions and the postnatal, to an ever-expanding cortex. The nuclear core and the cortex have different properties and can readily be physically separated. Communication and adhesion between the compartments is poor in older lenses. In vivo slit lamp examination reveals several zones of optical discontinuity in the lens. Different nomenclatures have been used to describe these, with the most common recognizing the embryonic, foetal, juvenile and adult nuclei as well as the cortex and outer cortex. Implicit in this nomenclature is the idea that the nuclear zones were generated at defined periods of development and growth. This review examines the relationship between the two compartments observed in vitro and the internal structures revealed by slit lamp photography. Defining the relationship is not as simple as it might seem because of remodeling and cell compaction which take place, mostly in the first 20 years of postnatal life. In addition, different investigators use different nomenclatures when describing the same regions of the lens. From a consideration of the dimensions, the dry mass contents and the protein distributions in the lens and in the various zones, it can be concluded that the juvenile nucleus and the layers contained within it, as well as most of the adult nucleus, were actually produced during prenatal life and the adult nucleus was completed within 3 months after birth, in the final stages of the prenatal growth mode. Further postnatal growth takes place entirely within the cortex. It can also be demonstrated that the in vitro nuclear core corresponds to the combined slit lamp nuclear zones. In view of the information presented in this review, the use of the terms foetal, juvenile and adult nucleus seems inappropriate and should be abandoned.

Localization of low molecular weight crystallin peptides in the aging human lens using a MALDI mass spectrometry imaging approach.

Low molecular weight (LMW) peptides, derived from the breakdown of the major eye lens proteins, the crystallins, accumulate in the human lens with age. These LMW peptides are associated with age-related lens opacity and cataract, with some shown to inhibit the chaperone activity of alpha-crystallin. However, the mechanism(s) giving rise to the production of these peptides, as well as their distribution within the lens, are not well understood. In this study, we have mapped the distribution of these crystallin-derived peptides present in human lenses of different ages using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Our data showed that most of these LMW peptides emerge in the lens at early middle-age, with peptides greater than 1778 Da in mass being confined to the water insoluble fractions, and to a lesser extent the water soluble fractions of older lenses. MALDI-IMS analyses showed that four peptides, derived from alphaA-, alphaB- and gammaS-crystallins, were confined to the lens nuclear fibre cells upon emergence during early middle-age, but were present in both the cortex and nucleus of old lenses. In contrast, another major peptide, derived from the C-terminal breakdown of betaA3-crystallin, was present in the cortical and nuclear regions of both young and old lenses. A comparison between age-matched cataractous and non-cataractous lenses showed no distinct differences in LMW peptide profiles, indicating that although cataract may be a potential consequence caused by the emergence of these peptides, it does not contribute directly to the peptide-generating process.

In vivo lens deficiency of the R49C alphaA-crystallin mutant.

The R49C mutation of alphaA-crystallin (alphaA-R49C) causes hereditary cataracts in humans; patients in a four-generation Caucasian family were found be heterozygous for this autosomal dominant mutation. We previously generated knock-in mouse models of this mutation and found that by 2 months of age, heterozygous mutant mice exhibited minor lens defects including reduced protein solubility, altered signaling in epithelial and fiber cells, and aberrant interactions between alphaA-crystallin and other lens proteins. In contrast, homozygous mutant alphaA-R49C knock-in mice displayed earlier and more extensive lens defects including small eyes and small lenses at birth, death of epithelial and fiber cells, and the formation of posterior, nuclear, and cortical cataracts in the first month of life. We have extended this study to now show that in alphaA-R49C homozygous mutant mice, epithelial cells failed to form normal equatorial bow regions and fiber cells continued to die as the mice aged, resulting in a complete loss of lenses and overall eye structure in mice older than 4 months. These results demonstrate that expression of the hereditary R49C mutant of alphaA-crystallin in vivo is sufficient to adversely affect lens growth, lens cell morphology, and eye function. The death of fiber cells caused by this mutation may ultimately lead to loss of retinal integrity and blindness.

Drug-induced lenticular opacity and accumulation of cholesterol-related substances in the lens cortex of dogs.

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.

The mechanisms of calcium homeostasis and signalling in the lens.

Excessive Ca(2+) can be detrimental to cells and raised levels of Ca(2+) in human lenses with cortical cataract have been found to play a major role in the opacification process. Ca(2+) homeostasis is therefore, recognised as having fundamental importance in lens pathophysiology. Furthermore, Ca(2+) plays a central role as a second messenger in cell signalling and mechanisms have evolved which give cells exquisite control over intracellular Ca(2+) ([Ca(2+)](i)) via an array of specialised regulatory and signalling proteins. In this review we discuss these mechanisms as they apply to the lens. Ca(2+) levels in human aqueous humour are approximately 1 mM and there is a large, 10,000 fold, inwardly directed gradient across the plasma membrane. In the face of such a large gradient highly efficient mechanisms are needed to maintain low [Ca(2+)](i). The Na(+)/Ca(2+) exchanger (NCX) and plasma membrane Ca(2+)-ATPase (PMCA) actively remove Ca(2+) from the cells, whereas the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) sequesters Ca(2+) in the endoplasmic reticulum (ER) Ca(2+) store. In lens epithelial cells the dominant role is played by the ATPases, whilst in the fibre cells NCX activity appears to be more important. Usually, [Ca(2+)](i) can be increased in a number of ways. Ca(2+) influx through the plasma membrane, for example, is mediated by an array of channels with evidence in the lens for the presence of voltage-operated Ca(2+) channels (VOCCs), receptor-operated Ca(2+) channels (ROCCs) and channels mediating store-operated Ca(2+) entry (SOCE). Ca(2+) signalling is initiated via activation of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTK) of which the lens expresses a surprisingly diverse array responding to various neurotransmitters, hormones, growth factors, autocoids and proteases. Downstream of plasma membrane receptors are IP(3)-gated channels (IP(3)Rs) and ryanodine receptors (RYRs) located in the ER, which when activated cause a rapid increase in [Ca(2+)](i) and these have also been identified in the lens. Through an appreciation of the diversity and complexity of the mechanisms involved in Ca(2+) homeostasis in normal lens cells we move closer to an understanding of the mechanisms which mediate pathological Ca(2+) overload as occurs in the process of cataract formation.