Autophagy and UPR in alpha-crystallin mutant knock-in mouse models of hereditary cataracts.

BACKGROUND: Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C+/- heterozygotes develop cataracts by 1-2months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens. METHODS: Wild-type, heterozygote (αA-R49C+/-), and homozygote (αA-R49C+/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting). RESULTS: Morphology was altered by embryonic day 17 in R49C+/+ lenses; R49C+/- lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G+/+ mice, suggesting its role in lens opacification. CONCLUSIONS: Mutated crystallins alter lens morphology, autophagy, and stress responses. GENERAL SIGNIFICANCE: Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Comparative proteomic analysis identifies age-dependent increases in the abundance of specific proteins after deletion of the small heat shock proteins αA- and αB-crystallin.

Mice with deletion of genes for small heat shock proteins αA- and αB-crystallin (αA/αB(-/-)) develop cataracts. We used proteomic analysis to identify lens proteins that change in abundance after deletion of these α-crystallin genes. Wild-type (WT) and αA/αB(-/-) knockout (DKO) mice were compared using two-dimensional difference gel electrophoresis and mass spectrometric analysis, and protein identifications were validated by Mascot proteomic software. The abundance of histones H2A, H4, and H2B fragment, and a low molecular weight ß1-catenin increased 2-3-fold in postnatal day 2 lenses of DKO lenses compared with WT lenses. Additional major increases were observed in abundance of ßB2-crystallin and vimentin in 30-day-old lenses of DKO animals compared with WT animals. Lenses of DKO mice were comprised of nine protein spots containing ßB2-crystallin at 10-40-fold higher abundance and three protein spots containing vimentin at ≥2-fold higher abundance than in WT lenses. Gel permeation chromatography identified a unique 328 kDa protein in DKO lenses, containing ß-crystallin, demonstrating aggregation of ß-crystallin in the absence of α-crystallins. Together, these changes provide biochemical evidence for possible functions of specific cell adhesion proteins, cytoskeletal proteins, and crystallins in lens opacities caused by the absence of the major chaperones, αA- and αB-crystallins.

p62 expression and autophagy in αB-crystallin R120G mutant knock-in mouse model of hereditary cataract.

The formation of cataracts is associated with the accumulation of protein aggregates in the ocular lens, suggesting that defective protein degradation plays a role in cataract pathogenesis. Accumulation of the p62 protein has recently been identified as a marker for impaired autophagy in a variety of tissues; however, little information exists on its expression in the ocular lens and in cataracts. In the present study we examined the expression of p62 in the mouse lens and compared its expression in wild-type lenses with that in lenses from knock-in mice with an arginine to glycine mutation in αB-crystallin (αB-R120G) that is known to cause human hereditary cataract. Immunohistochemical, immunoblotting, and transmission electron microscopic analyses of wild type and αB-R120G mutant mice were performed. To assess the effect of increased protein aggregation on autophagy, immunohistochemical staining was performed with an anti-p62 antibody, revealing the presence of p62-positive punctate staining in a band of denucleated cortical fiber cells. The number and size of p62 puncta were significantly greater in αB-R120G homozygous mutant lenses than in wild type and heterozygous mutant lenses. p62 staining was also abundant in lens epithelial cells and was concentrated around the nuclear membrane. Double-membraned structures similar to autophagosomes containing cellular cytoplasmic content were detected in lens epithelial cells by transmission electron microscopy. The autophagosomes in lens epithelial cells from αB-R120G homozygous mutant mice were larger than those in wild type mice. Double-membraned structures that are probably autophagosomes were also detected in cortical fiber cells and were more abundant in the αB-R120G homozygous mutant lens than the wild type lens. This study demonstrates p62 distribution as speckles in the lens fiber cells, altered levels of p62 expression, and the presence of autophagosomes in the ocular lens of αB-R120G mutant mice. We propose that autophagy is inhibited in the αB-R120G mutant lenses because of a defect in protein degradation after autophagosome formation. Further work is necessary to determine the relationship between αB-crystallin function, autophagy, and cataract formation.

Oxysterol Compounds in Mouse Mutant αA- and αB-Crystallin Lenses Can Improve the Optical Properties of the Lens.

Purpose: To investigate how cataract-linked mutations affect the gradient refractive index (GRIN) and lens opacification in mouse lenses and whether there is any effect on the optics of the lens from treatment with an oxysterol compound. Methods: A total of 35 mice including wild-type and knock-in mutants (Cryaa-R49C and Cryab-R120G) were used in these experiments: 26 mice were treated with topical VP1-001, an oxysterol, in one eye and vehicle in the other, and nine mice were untreated controls. Slit lamp biomicroscopy was used to analyze the lens in live animals and to provide apparent cataract grades. Refractive index in the lenses of 64 unfixed whole mouse eyes was calculated from measurements with X-ray phase tomography based on X-ray Talbot interferometry with a synchrotron radiation source. Results: Heterozygous Cryaa-R49C lenses had slightly irregularly shaped contours in the center of the GRIN and distinct disturbances of the gradient index at the anterior and posterior poles. Contours near the lens surface were denser in homozygous Cryab-R120G lenses. Treatment with topical VP1-001, an oxysterol, showed an improvement in refractive index profiles in 61% of lenses and this was supported by a reduction in apparent lens opacity grade by 1.0 in 46% of live mice. Conclusions: These results indicate that α-crystallin mutations alter the refractive index gradient of mouse lenses in distinct ways and suggest that topical treatment with VP1-001 may improve lens transparency and refractive index contours in some lenses with mutations.

Analysis of amyloid-like secondary structure in the Cryab-R120G knock-in mouse model of hereditary cataracts by two-dimensional infrared spectroscopy.

αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer's, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.

Activation of the unfolded protein response by a cataract-associated αA-crystallin mutation.

αA-crystallin is a lens chaperone that plays an essential role in the transparency and refractive properties of the lens. Mutations in αA-crystallin have been associated with the development of hereditary cataracts. The R49C mutation of αA-crystallin (αA-R49C) was identified in a four-generation Caucasian family with hereditary cataracts. The αA-R49C protein forms larger-than-normal oligomers in the lens and has decreased solubility. This aberrant αA-R49C oligomerization suggests that protein folding is altered. However, whether activation of the unfolded protein response (UPR) occurs during crystallin mutation-induced cataract formation and whether the UPR causes cell death under these conditions is unclear. We investigated UPR activation in an in vivo mouse model of αA-R49C using immunoblot analysis of lens extracts. We found that expression of the endoplasmic reticulum (ER) chaperone, BiP, was 5-fold higher in homozygous αA-R49C lenses than in wild type lenses. Analysis of proteins typically expressed during the UPR revealed that ATF-4 and CHOP levels were also higher in homozygous lenses than in wild type lenses, while the opposite was true of ATF-6 and XBP-1. Taken together, these findings show that mutation of αA-crystallin induces activation of the UPR during cataract formation. They also suggest that the UPR is an important mediator of cell death observed in homozygous αA-R49C lenses.

Quantitative biometric phenotype analysis in mouse lenses.

The disrupted morphology of lenses in mouse models for cataracts precludes accurate in vitro assessment of lens growth by weight. To overcome this limitation, we developed morphometric methods to assess defects in eye lens growth and shape in mice expressing the alphaA-crystallin R49C (alphaA-R49C) mutation. Our morphometric methods determine quantitative shape and dry weight of the whole lens from histological sections of the lens. This method was then used to quantitatively compare the biometric growth patterns of lenses of different genotypes of mice from birth to 12 months. The wild type dry lens weights determined using the morphometric method were comparable to previously reported weights. Next we applied the method to assessing the lenses of alphaA-R49C knock-in mice, which exhibit decreased alphaA-crystallin protein solubility, resulting in a variety of growth abnormalities including early cataract formation, decreased eye and lens size, failure to form the equatorial bow region, and continued lens cell death, sometimes resulting in the entire loss of the lens and eye. Our morphometric methods reproducibly quantified these defects by combining histology, microscopy, and image analysis. The volume measurement accurately represented the total growth of the lens, whereas the geometric shape of the lens more accurately quantified the differences between the growth of the mutant and wild-type lenses. These methods are robust tools for measuring dry lens weight and quantitatively comparing the growth of small lenses that are difficult to weigh accurately such as those from very young mice and mice with developmental lens defects.

αA-Crystallin associates with α6 integrin receptor complexes and regulates cellular signaling.

α-Crystallins are small heat-shock proteins important to lens transparency that provide the lens with its refractive properties. In their role as molecular chaperones, these crystallins also prevent protein aggregation, affect cytoskeletal remodeling, enhance resistance to cell stress, and provide lens cells with protection against apoptosis. While many of the functions assigned to αA-crystallin are attributable to its presence in the cytoplasm of lens cells, αA-crystallin also has been detected at the lens plasma membrane. However, how αA-crystallin becomes linked to the plasma membrane or what its functions are at this site has remained unknown. In this study, we examined the mechanisms by which αA-crystallin becomes associated with the lens membrane, focusing specifically on its interaction with membrane receptors, and the differentiation-specificity of these interactions. We also determined how the long-term absence of αA-crystallin alters receptor-linked signaling pathways. αA-crystallin association with membrane receptors was determined by co-immunoprecipitation analysis; its membrane localization was examined by confocal imaging; and the effect of αA-crystallin loss-of-function on the activation state of signaling molecules in pathways linked to membrane receptors was determined by immunoblot analysis. The results show that, in lens epithelial cells, plasma membrane αA-crystallin was primarily localized to apicolateral borders, reflecting the association of αA-crystallin with E-cadherin complexes. These studies also provide the first evidence that αA-crystallin maintained its association with the plasma membrane in lens cortical fiber cells, where it was localized to lateral interfaces, and further show that this association was mediated, in part, by αA-crystallin interaction with α6 integrin receptor complexes. We report that the absence of αA-crystallin led to constitutive activation of the stress kinases p38 and JNK, classical inducers of apoptotic cell death, and the loss of the phospho-Bad pro-survival signal, effects that were greatest in differentiating lens fiber cells. Concurrent with this, activation of FAK and ERK kinases was increased, demonstrating that these receptor-linked pathways also were dysregulated in the absence of αA-crystallin. These data link αA-crystallin plasma membrane association to its differentiation-state-specific interaction with E-cadherin and α6 integrin receptor complexes. The changes in cell signaling in αA-crystallin-null lenses suggest that dysregulation of receptor-linked cell-signaling pathways that accompany the failure of αA-crystallin to associate with membrane receptors may be responsible for the induction of apoptosis. The observed changes in lens cell signaling likely reflect long-term functional adaptations to the absence of the αA-crystallin chaperone/small heat-shock protein.

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.

Creatine kinase/α-crystallin interaction functions in cataract development.

Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the αA-crystallin (cryaa) and αB-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with α-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and α-crystallin, we investigated whether α-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between α-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and α-crystallin, suggesting that the interaction between CK and α-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble α-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and α-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between α-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

Alpha-crystallin mutations alter lens metabolites in mouse models of human cataracts.

Cataracts are a major cause of blindness worldwide and commonly occur in individuals over 70 years old. Cataracts can also appear earlier in life due to genetic mutations. The lens proteins, αA- and αB-crystallins, are chaperone proteins that have important roles maintaining protein solubility to prevent cataract formation. Mutations in the CRYAA and CRYAB crystallin genes are associated with autosomal dominant early onset human cataracts. Although studies about the proteomic and genomic changes that occur in cataracts have been reported, metabolomics studies are very limited. Here, we directly investigated cataract metabolism using gas-chromatography-mass spectrometry (GC-MS) to analyze the metabolites in adult Cryaa-R49C and Cryab-R120G knock-in mouse lenses. The most abundant metabolites were myo-inositol, L-(+)-lactic acid, cholesterol, phosphate, glycerol phosphate, palmitic and 9-octadecenoic acids, α-D-mannopyranose, and ß-D-glucopyranose. Cryaa-R49C knock-in mouse lenses had a significant decrease in the number of sugars and minor sterols, which occurred in concert with an increase in lactic acid. Cholesterol composition was unchanged. In contrast, Cryab-R120G knock-in lenses exhibited increased total amino acid content including valine, alanine, serine, leucine, isoleucine, glycine, and aspartic acid. Minor sterols, including cholest-7-en-3-ol and glycerol phosphate were decreased. These studies indicate that lenses from Cryaa-R49C and Cryab-R120G knock-in mice, which are models for human cataracts, have unique amino acid and metabolite profiles.

Changes in relative histone abundance and heterochromatin in αA-crystallin and αB-crystallin knock-in mutant mouse lenses.

OBJECTIVE: Understanding the mechanisms of cataract formation is important for age-related and hereditary cataracts caused by mutations in lens protein genes. Lens proteins of the crystallin gene families α-, ß-, and γ-crystallin are the most abundant proteins in the lens. Single point mutations in crystallin genes cause autosomal dominant cataracts in multigenerational families. Our previous proteomic and RNAseq studies identified genes and proteins altered in the early stages of cataract formation in mouse models. Histones H2A, H2B, and H4 increase in abundance in αA- and αB-crystallin mutant mouse lenses and in cultured cells expressing the mutant form of αA-crystallin linked with hereditary cataracts. RESULTS: In this study of histones in mutant lenses, we extracted histones from adult mouse lenses from cryaa-R49C and cryab-R120G mutant knock-in mice. We characterized the histones using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometric analysis and gel electrophoresis and characterized the lens nucleus morphology using electron microscopy (EM). The relative abundance of histone H3 protein decreased in lenses from cryaa-R49C mutant mice and the relative abundance of histone H2 increased in these lenses. Electron microscopy of nuclei from cryaa-R49C-homozygous mutant mouse lenses revealed a pronounced alteration in the distribution of heterochromatin.

AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

BACKGROUND: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family. METHODS: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared. RESULTS: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein. CONCLUSION: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

Mechanism of Action of VP1-001 in cryAB(R120G)-Associated and Age-Related Cataracts.

Purpose: We previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity. Methods: We compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy. Results: Docking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology. Conclusions: The ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts.

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.

The lens epithelium: focus on the expression and function of the alpha-crystallin chaperones.

Lens epithelial cells are the parental cells responsible for growth and development of the transparent ocular lens. Many elegant investigations into their biology have focused on the factors that initiate and regulate lens epithelial cell differentiation. Because they serve key transport and cell maintenance functions throughout life, and are the primary source of metabolic activity in the lens, mechanisms to maintain lens epithelial cell integrity and survival are critical for lens transparency. The molecular chaperones alpha-crystallins are abundant proteins synthesized in the differentiated lens fiber cell cytoplasm. However, their expression in lens epithelial cells has only been appreciated very recently. Besides their important roles in the refractive and light focusing properties of the lens, alpha-crystallins have been implicated in a number of non-refractive pathways including those involving stress response, apoptosis and cell survival. The most convincing evidence for their importance in the lens epithelium has been shown by studies on the properties of lens epithelial cells from alphaA and alphaB-crystallin gene knockout mice. Novel combination of genetics, cell and molecular biology should lead to a greater understanding of how lens epithelial cells proliferate, differentiate and survive.

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.

In vitro interactions of histones and α-crystallin.

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone-α-crystallin binding with a Kd of 4 × 10-7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone-α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone-α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.

Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract.

The mammalian eye lens expresses a high concentration of crystallins (α, ß and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.

Up-regulation of tau, a brain microtubule-associated protein, in lens cortical fractions of aged alphaA-, alphaB-, and alphaA/B-crystallin knockout mice.

PURPOSE: Alpha-crystallin is expressed at high levels in the lens in a complex of alphaA- and alphaB-crystallin subunits in 3:1 molar ratios, and is known to maintain the solubility of unpolymerized tubulin and enhance the resistance of microtubules to depolymerization, but its effect on proteins classically associated with microtubule stability (microtubule associated proteins) in the lens is unknown. In the present study we examined the expression of the brain microtubule associated protein tau in lenses of alpha-crystallin gene knockout mice. METHODS: Quantitative RT-PCR, immunoblotting, cryo-immunoelectron microscopic and immunohistochemical methods were used to characterize the expression of tau in the lenses of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mice. RESULTS: Immunoreactivity to tau, a 45-66 kDa brain microtubule associated protein that has been best characterized in neurons and neuronal pathologies, was uniquely upregulated in lens cortical fiber cells with aging and was associated with the microtubule fraction of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mouse lenses, but was undetectable in wild type lenses. Quantitative RT-PCR analysis further showed an upregulation of tau transcripts in alphaA(-/-)- and alphaA/B(-/-)-crystallin lenses. Brain microtubule fractions served as a positive control for tau in these experiments. An increase in phosphorylation of tau was detected in alphaA(-/-)- and alphaB(-/-)-crystallin brain proteins. CONCLUSIONS: Although tau aggregation and alphaB-crystallin expression have been shown to increase in neurodegenerative diseases, surprisingly tau expression increases in the alpha-crystallin knockout lenses, suggesting that alphaA- and alphaB-crystallins are potentially important regulators of tau expression in lens.