Usha P. Andley

Crystallins in the eye: Function and pathology.

Crystallins are the predominant structural proteins in the lens that are evolutionarily related to stress proteins. They were first discovered outside the vertebrate eye lens by Bhat and colleagues in 1989 who found alphaB-crystallin expression in the retina, heart, skeletal muscles, skin, brain and other tissues. With the advent of microarray and proteome analysis, there is a clearer demonstration that crystallins are prominent proteins both in the normal retina and in retinal pathologies, emphasizing the importance of understanding crystallin functions outside of the lens. There are two main crystallin gene families: alpha-crystallins, and betagamma-crystallins. alpha-crystallins are molecular chaperones that prevent aberrant protein interactions. The chaperone properties of alpha-crystallin are thought to allow the lens to tolerate aging-induced deterioration of the lens proteins without showing signs of cataracts until older age. alpha-crystallins not only possess chaperone-like activity in vitro, but can also remodel and protect the cytoskeleton, inhibit apoptosis, and enhance the resistance of cells to stress. Recent advances in the field of structure-function relationships of alpha-crystallins have provided the first clues to their underlying roles in tissues outside the lens. Proteins of the betagamma-crystallin family have been suggested to affect lens development, and are also expressed in tissues outside the lens. The goal of this paper is to highlight recent work with lens epithelial cells from alphaA- and alphaB-crystallin knockout mice. The use of lens epithelial cells suggests that crystallins have important cellular functions in the lens epithelium and not just the lens fiber cells as previously thought. These studies may be directly relevant to understanding the general cellular functions of crystallins.

Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

αA- and αB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of αA- and αB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of αA- and αB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of αA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of αB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor α. To further study the relative protective activities of αA- and αB-crystallins, we created a cell line derived from αA−/−αB−/− double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line αAαBKO1 derived from αA/αB double knockout cells was transfected with αA- or αB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either αA-crystallin or αB-crystallin were isolated. The ability of αA- or αB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of αA-crystallin was two to three-fold higher than that of αB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of αA- and αB-crystallin. Since the expression of αA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.

The Molecular Chaperone αA-Crystallin Enhances Lens Epithelial Cell Growth and Resistance to UVA Stress

αA-Crystallin (αA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of αA, but in differentiated fiber cells, αA is the most abundant soluble protein. The lenses of αA-knock-out mice develop opacities at an early age, implying a critical role for αA in the maintenance of fiber cell transparency. However, the function of α-crystallin in the lens epithelium is unknown. To investigate the physiological function of αA in lens epithelial cells, we used the following two systems: αA knock-out (αA(−/−)) mouse lens epithelial cells and human lens epithelial cells that overexpress αA. The growth rate of αA(−/−) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of αA(−/−) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the αA(−/−) cultures was increased 40-fold over wild type. In cells lacking αA, UVA exposure modified F-actin, but actin was protected in cells expressing αA. Stably transfected cell lines overexpressing human αA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoαA. Cells overexpressing αA were resistant to UVA stress, as determined by clonogenic survival. αA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of αA was not associated with its relocalization to the nucleus. These results indicate that αA has important cellular functions in the lens over and above its well characterized role in refraction.

CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α‐, β‐, and γ‐, was studied by using fluorescence and circular dichroism techniques. α‐Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α‐crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H2O2‐mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α‐crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.

The R116C Mutation in αA-crystallin Diminishes Its Protective Ability against Stress-induced Lens Epithelial Cell Apoptosis

αA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of αA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in αA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C αA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C αA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the αA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type αA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4- to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. F-actin was protected in UVA-treated cells expressing the wild type αA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant αA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.

Photodamage to the eye

Photodamage to the eye involves both cumulative damage to ocular tissues with ambient solar or manmade sources of radiation, as well as acute exposures in unnaturally bright environments. Photochemical damage by acute exposures, i.e. corneal damage by UV in photokeratitis or snowblindness, lens opacification by acute UV-B exposures and retinal damage from sungazing or solar retinitis, is well documented. Since the corneal epithelium is a highly regenerative tissue, and sustained damage is incurred infrequently, investigations of photodamage to lens and retina have received much more attention. The important question is, whether or not chronic exposure to optical radiation under normal sunlight or other artificial illumination can cause an opacification of the lens or damage the retina, comparable to that occurring under conditions of unnaturally high light exposures. Various lines of evidence suggest that cumulative damage to the macromolecules of the lens and the retina, by photochemical generation of active species of oxygen, can account for some of the changes observed in these tissues in the diseased state. Photodamage to the tissues along the optic axis of the eye, namely the cornea, aqueous humor, lens, vitreous, retina and retinal pigment epithelium, is primarily caused by the wavelengths of radiation absorbed by each tissue. The cornea absorbs wavelengths below 295 nm, and transmits all longer wavelength radiation to the aqueous and lens. The lens contains chromophores which absorb between 295-400 nm, and transmit wavelengths longer than 400 nm to the retina. The absorption of the normal lens increases with age, due to a progressive accumulation of age-related chromophores (Lerman, 1987) thus providing an effective filter of near-UV and short wavelength visible light for the retina. The research in this field during the past two decades has been included in the book, Optical Radiation and Visual Health (Waxler and Hitchins, 1986). Other reviews are Dayhaw-Barker and

Pharmacological chaperone for α-crystallin partially restores transparency in cataract models

A visionary approach to transparency Cataracts are the most common cause of vision loss, especially in our ever-increasing elderly population. Cataracts arise when crystallin, a major protein component of the eye lens, begins to aggregate, which causes the lens to become cloudy. Makley et al. explored whether small molecules that reverse this aggregation might have therapeutic potential for treating cataracts, which normally require surgery (see the Perspective by Quinlan). They used a screening method that monitors the effect of ligands on temperature-dependent protein unfolding and identified several compounds that bind and stabilize the soluble form of crystallin. In proof-of-concept studies, one of these compounds improved lens transparency in mice. Science, this issue p. 674; see also p. 636 A compound that reverses the molecular cause of cataract formation improves eye lens transparency in mice. [Also see Perspective by Quinlan] Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

Reduced survival of lens epithelial cells in the alphaA-crystallin-knockout mouse

αA-Crystallin (αA) is a molecular chaperone expressed preferentially in the lens. αA transcripts are first detected during the early stages of lens development and its synthesis continues as the lens grows throughout life. αA–/– mouse lenses are smaller than controls, and lens epithelial cells derived from these mice have diminished growth in culture. In the current work, we tested the hypothesis thatαA prevents cell death at a specific stage of the cell cycle in vivo. Seven-day-old 129Sv (wild-type) and αA–/– mice were injected with 5-bromo-2′-deoxyuridine (BrdU) to label newly synthesized DNA in proliferating cells. To follow the fate of the labeled cells, wholemounts of the capsule epithelial explants were made at successive times after the BrdU pulse, and the labeling index was determined. Immunofluorescence and confocal microscopy showed that both wild-type andα A–/– cells had a 3-hour labeling index of 4.5% in the central region of the wholemount, indicating that the number of cells in S phase was the same. Twenty-four hours after the pulse, individual cells labeled with BrdU had divided and BrdU-labeled cells were detected in pairs. The 24-hour labeling index in the wild-type lens was 8.6%, but in theα A–/– lens it was significantly lower, suggesting that some of the cells failed to divide and/or that the daughter cells died during mitosis. TUNEL labeling was rarely detected in the wild-type lens, but was significant and always detected in pairs in theα A–/– wholemounts. Dual labeling with TUNEL and BrdU also suggested that the labeled cells were dying in pairs in theα A–/– lens epithelium. Immunolabeling of wholemounts with β-tubulin antibodies indicated that the anaphase spindle in a significant proportion of αA–/– cells was not well organized. Examination of the cellular distribution of αA in cultured lens epithelial cells showed that it was concentrated in the intercellular microtubules of cells undergoing cytokinesis. These data suggest that αA expression in vivo protects against cell death during mitosis in the lens epithelium, and the smaller size of theα A–/– lens may be due to a decrease in the net production of epithelial cells.

Difference in Phototoxicity of Cyclodextrin Complexed Fullerene [(γ-CyD)2/C60] and Its Aggregated Derivatives toward Human Lens Epithelial Cells

The water-soluble fullerene derivative gamma-cyclodextrin bicapped C(60) [(gamma-CyD)(2)/C(60), CDF0] has several clinical applications, including use as a drug carrier to bypass the blood ocular barriers or a photosensitizer to treat tumors in photodynamic therapy. We have assessed the potential ocular toxicity of (gamma-CyD)(2)/C(60) and its aggregated derivatives induced by UVA and visible light in vitro in human lens epithelial cells (HLE B-3). Cell viability using the MTS assay demonstrated that 2 microM (gamma-CyD)(2)/C(60) was highly phototoxic to HLE B-3 cells with UVA irradiation, while no effect was observed in the presence of visible light or when maintained in the dark. In contrast, the aggregated derivative (CDF150) showed neither cytotoxicity nor any phototoxic effect even at 30 microM with either UVA or visible light irradiation. In lens cells treated with (gamma-CyD)(2)/C(60), phototoxicity was manifested as apoptosis. Singlet oxygen production measurement using the EPR/TEMP trapping technique determined that (gamma-CyD)(2)/C(60) (CDF0) efficiently produced singlet oxygen. The rate of singlet oxygen production decreased with increased aggregation, with no production by the fully aggregated sample formed after 150 min of heating (CDF150). UVA irradiation of HLE B-3 in the presence of (gamma-CyD)(2)/C(60) resulted in a significant rise in intracellular protein-derived peroxides. The singlet oxygen quenchers sodium azide and histidine each significantly protected lens cells against (gamma-CyD)(2)/C(60) photodamage, but lutein and Trolox (vitamin E) did not. Clearly, singlet oxygen is an important intermediate in the phototoxicity of monomeric (gamma-CyD)(2)/fullerene. Our results also demonstrate that UVA-blocking sunglasses can limit the ocular phototoxicity of this nanomaterial, while nontoxic endogenous antioxidants like lutein or Trolox cannot provide adequate protection.

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

β‐Crystallins (β1,‐, β2‐ and β3‐crystallin) comprise nearly half the protein of the human lens. The effect of near‐UV radiation, which is one of the possible risk factors in cataract formation, on the p‐crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (λcx 340 nm, λcm 400–600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H202 in the irradiated human β‐crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O‐2 anion; the latter spontaneously dismutates to H202, presumably via O ‐2 ‐ protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of β1,‐, β2‐ and β3‐crystallins from human lenses of different age.