Dense Nuclear Cataract Caused by the B-Crystallin S11R Point Mutation

Dense Nuclear Cataract Caused by the ␥B-Crystallin S11R Point Mutation

Lin Li,1 Bo Chang,2 Catherine Cheng,3 Da Chang,2 Norman L. Hawes,2 Chun-hong Xia,1 and Xiaohua Gong1,3

PURPOSE. To identify the causative gene mutation for a new are able to synthesize proteins or metabolites that maintain the dominant cataract in mice and to investigate the molecular homeostasis of organelle-free inner fiber cells. Several studies basis for how the mutated gene leads to a dense nuclear have begun to unravel some insights about fiber cell matura- cataract. tion.2–4 ␣ ␤ ␥ METHODS. Genomewide linkage analysis and DNA sequencing Crystallin proteins ( , , and classes) account for more than 90% of total lens proteins.5 Lens transparency is suggested were used to determine the gene mutation. Histology, immu- 6 nohistochemistry, and Western blotting were used to charac- to rely on a short-range order of lens crystallin proteins. It is terize lens phenotypes. Ion concentrations were measured by speculated that different crystallin isoforms are needed for the an inductively coupled plasma–optical emission spectrometer appropriate arrangement of the interfaces of membrane and (ICP-OES). cytoskeleton in lens fiber cells to ensure transparency and a high refractive index. ␣-Crystallins, consisting of ␣A and ␣B RESULTS. A point mutation (A to C) of the ␥B-crystallin gene, ␥ subunits, are members of the small heat shock protein family which results in the B-S11R mutant protein, was identified in and have chaperone-like activity that prevents the aggregation this cataractous mouse line. Homozygous mutant mice devel- of denatured proteins in vitro.7–9 Both ␤- and ␥-crystallins are oped dense nuclear cataracts associated with disrupted inner ␥ structural proteins that share a common Greek motif and that lens fiber cells. Immunohistochemistry data revealed -crystal- are extremely heat stable.10 ␤-Crystallins form dimers and lin aggregates at the cell boundaries of inner mature fibers that higher oligomers, whereas ␥-crystallins exist as monomers in lose actin filaments. Western blotting showed an increased the lens. Different ␥-crystallin isoforms account for approxi- degradation of crystallin proteins correlated with the nuclear mately one third of total lens proteins.5 Identification of ␥N- cataract. ICP-OES confirmed a substantial elevation of calcium crystallin suggests an evolutionary link between ␤- and concentration in mutant lenses. ␥-crystallins.11 It is unknown whether ␤- and ␥-crystallins CONCLUSIONS. This dominant cataract was caused by the ␥B- play active roles in various cellular processes during differ- S11R mutation. Mutant ␥B-S11R proteins triggered the ␥-crys- entiation and maturation of lens fiber cells. However, it is tallin aggregation that probably disrupted membrane-cytoskel- important to note that these crystallins are expressed in eton structures of inner fiber cells, causing increased calcium tissues other than the lens, suggesting that they may have influxes. Subsequent activation of calcium-dependent protein other functions.12 degradation and degeneration of inner mature fiber cells led to ␥-Crystallin isoforms A-F show approximately 77% to 97% the dense nuclear cataract. (Invest Ophthalmol Vis Sci. 2008; identity at the protein level.11 Studies of recombinant, native, 49:304–309) DOI:10.1167/iovs.07-0942 and mutant ␥-crystallins have demonstrated significant varia- tion in phase separation of different ␥-crystallin isoforms and he eye lens is composed of lens cells wrapped by a colla- have provided some insights to explain how different ␥-crys- Tgen-based membrane, known as the lens capsule. Lens tallin isoforms contribute to “cold cataracts” induced by lower cells are predominantly elongated fiber cells covered by a temperature.13–16 Mutations that affect the stability and solu- monolayer of epithelial cells at the surface of the anterior bility of ␥-crystallins can lead to cataracts in humans and hemisphere.1 Lens inner fiber cells are mature fibers that lack mice,17,18 and some new studies suggest that ␥-crystallins may intracellular organelles to minimize light scattering. Thus, only have other important roles in the lens besides their role as anterior surface epithelial cells and newly differentiated fiber passive structural components.12,15 cells in the lens periphery contain intracellular organelles, such Here we report a severe nuclear cataract caused by a new as endoplasmic reticulum, Golgi, and mitochondria; those cells point mutation of ␥B-crystallin. We have found that this mutation specifically altered the subcellular distribution of ␥-crystallin in mature fiber cells and led to unique cellular and From the 1Vision Science Program and School of Optometry, biochemical changes that resulted in a dense nuclear cataract. University of California, Berkeley, Berkeley, California; 2The Jackson This work provides some new information about how ␥B- Laboratory, Bar Harbor, Maine; and 3UC Berkeley/UCSF Joint Graduate crystallin is needed for the appropriate subcellular arrange- Program in Bioengineering, University of California, Berkeley, Berke- ment of proteins in lens mature fiber cells. ley, California. Supported by National Eye Institute Grants EY013849 and EY007758. Submitted for publication July 24, 2007; revised September 14, MATERIALS AND METHODS 2007; accepted November 14, 2007. Disclosure: L. Li, None; B. Chang, None; C. Cheng, None; D. Chang, None; N.L. Hawes, None; C. Xia, None; X. Gong, None Mouse Breeding and Causative The publication costs of this article were defrayed in part by page Gene Identification charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Mouse care and breeding were performed according to the Animal Corresponding author: Xiaohua Gong, Vision Science Program Care and Use Committee (ACUC)-approved animal protocol (UC Berke- and School of Optometry, University of California, Berkeley, 693 Minor ley) and the ARVO Statement for the Use of Animals in Ophthalmic and Hall, Vision Science Program and School of Optometry, Berkeley, CA Vision Research. Mouse pupils were dilated with 1% atropine and 1% 94720-2020; [email protected]. phenylephrine before the eyes were examined for lens clarity by a slit

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lamp. Primers for different ␥-crystallin genes were described previously for the ␥B-I4F gene mutation.17

Histology, Immunohistochemistry, and Western Blotting Histology. Enucleated eyeballs opened at the anterior chamber or posterior vitreous were immersed in a ﬁxative solution containing 2% glutaraldehyde and 2.5% formaldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature for 5 days. Samples were postﬁxed in 1%

aqueous OsO4 and then dehydrated through graded acetone. Samples were embedded in eponate 12-araldyte 502 resin (Ted Pella, Redding, CA). Lens sections (1 ␮m thick) across the equatorial plane were collected on glass slides and stained with toluidine-blue. Bright-field images were acquired through a light microscope (Axiovert 200; Carl Zeiss, Oberkochen, Germany) with a digital camera. Immunohistochemistry. A previously described procedure was used for preparing lens cryosections for immunohistochemical staining.19 A laser confocal microscope (Leica, Wetzlar, Germany) was used to collect fluorescent images. Western Blotting. Lens total proteins were prepared by homog- enizing enucleated fresh lenses that were weighed and homogenized directly in the sample buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol, and 0.001% bromophenol blue). Equal volumes (20 ␮L) of samples were loaded on a 12.5% SDS-PAGE gel for separation, and separated proteins were transferred to a poly- vinylidene difluoride membrane (Bio-Rad, Hercules, CA). Lens crystallin proteins were detected by Western blotting with rabbit polyclonal FIGURE 1. A dominant nuclear cataract linked to the ␥B-S11R muta- antibodies against ␣- and ␥-crystallins (generously provided by Joseph tion. (A) Lens pictures of wild-type, heterozygous (heter), and homozy- Horwitz at University of California at Los Angles), ␤-crystallin (gener- gous (homo) mutant mice at P1, P7, and P21. Images were taken of ously provided by J. Samuel Zigler at the National Eye Institute), and a enucleated fresh lenses immersed in PBS at 37°C. Scale bar, 1 mm. (B) DNA sequencing data verified an A-to-C mutation in the Crygb gene. mouse monoclonal antibody against ␤-actin (Sigma, St. Louis, MO). This point mutation resulted in a substitution of the serine residue by More than three sets of lens protein samples of different mice were an arginine at the 11th codon of the ␥B-crystallin protein (␥B-S11R). examined, and representative data were shown. Ion Concentration Measurement grounds, including C57BL/6J or 129 strains. Fifty-four back- Lens ion concentration was measured by inductively coupled plasma– cross mice between Nm3062 mutant and wild-type CAST/Ei optical emission spectrometry (ICP-OES) from a core facility at the mice underwent phenotyping and genotyping for genomewide University of California, San Diego. The method was described previ- linkage analysis. This mutation was mapped to a region be- ously.20 Lenses were dissected from postnatal day (P)10 wild-type, tween the D1Mit19 and D1Mit21 markers on mouse chromo- heterozygous and homozygous mutant mice and then immediately some 1. A cluster of ␥-crystallin genes (CrygA-F) was located in subjected to vacuum drying for 48 hours. Dry lenses were weighed and this region. DNA sequence analysis of these ␥-crystallin genes solubilized in 500 ␮L nitric acid (33.5 ϳ 35%; Fisher Scientific, Pitts- verified an A-to-C point mutation of the 11th codon of Crygb, burgh, PA) for 12 hours at 37°C with shaking and then diluted with which resulted in a substitution of serine with arginine at that water into 3 mL. Samples were further diluted to reach ion concentra- codon in ␥B-crystallin (␥B-S11R; Fig. 1B). No mutation was tions of 20 ppb to 1 ppm for measurement. According to the estimated detected in other ␥-crystallin genes. sample ion concentration, a series of standards was made. Ion concentrations were determined by their intensities acquired by the instru- Normal Peripheral Fiber Cells and Disintegrated ment. Measurement error was approximately 3%. Ion concentrations Inner Fiber Cells in the ␥B-S11R Mutant Lenses were normalized by lens dry weight. Toluidine blue-stained lens histology sections were prepared from neonatal mice (Fig. 2). These histologic sections showed RESULTS that peripheral fibers developed normally at the bow region of A Dominant Nuclear Cataract Caused by the P3 mutant lenses. However, inner fiber cells of heterozygous ␥ ␥B(S11R/ϩ) and homozygous ␥B(S11R/S11R) lenses displayed B-S11R Mutation uneven toluidine blue staining with abnormal darkly stained We identified a dominant cataract from a spontaneous mouse areas. Disintegrated fiber cells appeared only in the core of mutant line (Nm3062) in the A/J strain background by slit lamp homozygous ␥B(S11R/S11R) lenses, whereas lens peripheral examination. Heterozygous mice displayed hazy nuclear cata- fiber cells displayed relatively normal morphology. racts, whereas homozygous mice developed dense nuclear The periphery of ␥B(S11R/S11R) mutant lenses remained cataracts at weaning age (Fig. 1A). Further examination of transparent even in older mice (Fig. 1A). Representative histo- enucleated fresh lenses revealed that this nuclear cataract logic data of lens peripheral fibers between wild-type and could be obviously visualized in mutant mice at P7, but not at ␥B(S11R/S11R) mutant mice at P21 were shown (Fig. 2B). Lens P1. Mutant lenses also displayed a unique ringlike structure— cross-sections revealed that peripheral fiber cells of P21 located at approximately 220 to 350 ␮m away from the lens ␥B(S11R/S11R) lenses remained relatively normal, similar to capsule—that caused abnormal light scattering. Homozygous those of P21 wild-type lenses. Therefore, histologic data con- mutant mice developed dense nuclear cataracts with full pen- firmed that altered inner fiber cells were correlated to the etrance in the A/J strain background or in mixed strain back- nuclear cataract phenotype caused by the ␥B-S11R mutation.

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Nuclear Cataracts and the Degradation of Crystallin Proteins Morphologic data suggest that dense nuclear cataracts are associated with disintegrated inner ﬁber cells. To determine whether protein degradation occurred in these disintegrated lens ﬁber cells, we used Western blotting to analyze crystallins in wild-type and mutant lenses. Cleaved forms of different crystallins (␣A, ␣B, ␤, ␥) were detected in P21 mutant lenses but not in wild-type lenses. Moreover, cleaved ␣B- and ␥-crystallins were detected in P7 mutant lenses (Fig. 4). Homozygous mutant lenses contained more cleaved crystallins than heterozygous mutant lenses. There was no detectable cleavage of ␣B-crystallin in P1 mutant lenses (data not shown). Thus, these Western blotting results indicated that the severity of the nuclear cataract was associated with the amount of cleaved crystallins (␣ and ␤/␥) in mutant lenses.

FIGURE 2. Histology of P3 and P21 wild-type and ␥B-S11R mutant lenses. (A, left) P3 whole lens sections. Scale bar, 100 ␮m. Right: high-magnification images of lens bow and inner regions. Bow regions of wild-type (WT) and mutant lenses displayed normal morphology. However, inner regions of ␥B(S11R/ϩ) and ␥B(S11R/S11R) lenses displayed uneven staining (arrowheads), and only ␥B(S11R/S11R) lenses revealed disintegrated fiber cells (arrow). (B) Lens cross-sections revealed that peripheral fiber cells of P21 ␥B(S11R/S11R) lenses remained relatively normal, similar to those of P21 wild-type lenses. Scale bars: (A)10␮m; (B)20␮m.

The Aggregation of ␥-Crystallins Adjacent to the Cell Boundary and the Absence of F-Actin in Inner Fiber Cells Based on the fact that the ␥B-S11R mutation leads to a dense nuclear cataract different from the lamellar cataract caused by the ␥B-I4F mutation, as reported previously,17 we hypothesize that, unlike ␥B-I4F, ␥〉-S11R mutant proteins trigger a unique event to disrupt inner fiber cells to cause nuclear cataracts. To evaluate this hypothesis and to understand how mutant proteins lead to the disruption of inner fiber cells in ␥B(S11R/ S11R) lenses, we examined the cellular distribution of ␥-crystallins in lens fiber cells of wild-type and mutant lenses. ␥-Crystallins account for approximately one third of total proteins in lens fibers. Immunostaining of ␥-crystallin revealed uniformly distributed signal in peripheral and interior fiber cells of wild-type lenses (Fig. 3A). ␥-Crystallins were also uniformly stained in peripheral fiber cells of ␥B(S11R/S11R) mutant lenses. However, ␥-crystallin aggregates were detected adjacent to the cell boundary of inner fibers and were segre- FIGURE 3. Immunostaining of ␥-crystallins and actin filaments in lens gated from actin filaments (Fig. 3A). Further studies of P7 and fiber cells. (A) P1 wild-type and homozygous ␥B(S11R/S11R) lens P21 ␥B(S11R/S11R) lenses revealed that actin filaments com- frozen sections were stained with anti–␥-crystallin antibody (green) pletely disappeared in inner fiber cells, starting at approxi- and rhodamine-phalloidin (red). Boxes: selected areas that show sep- mately 220 to 350 ␮m away from the lens capsule (Fig. 3B). arated and merged fluorescent images of ␥-crystallin and F-actin in lens Thus, these results indicate that ␥B-S11R mutant proteins lead inner fiber cells. White arrows: ␥-Crystallin aggregates adjacent to cell to the formation of unique ␥-crystallin aggregates and the boundaries. White arrowheads: F-actin. (B) Rhodamine-phalloidin– stained frozen sections of P7 and P21 wild-type and ␥B(S11R/S11R) disruption of actin filaments in inner fiber cells. The loss of lenses. Actin filaments disappeared in inner fiber cells of ␥B(S11R/ F-actin staining in the lens section (Fig. 3B) seems to be cor- S11R) mutant lenses, starting at approximately 350 ␮m from the lens related with the appearance of the abnormal ring, peripheral to capsule in the P7 sample and approximately 200 ␮m in the P21 sample the dense nuclear cataract in ␥B(S11R/S11R) lenses (Fig. 1A). (white arrowheads). Scale bars, 50 ␮m.

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Elevated Calcium Concentration in Mutant Lenses Activation of calcium-dependent proteases in mouse lenses often results in crystallin degradation. An influx of extracellular calcium into inner fiber cells could activate calcium-dependent proteases. We hypothesized that elevated calcium levels lead to increased crystallin degradation, resulting in severe nuclear cataracts in ␥B(S11R/S11R) mutant lenses. Therefore, we examined the total amount of ions, including calcium, magnesium, sodium, and potassium, in lenses by ICP-OES. The total calcium level was increased approximately fourfold in ␥B(S11R/S11R) lenses compared with wild-type lenses, whereas sodium, magnesium, and potassium levels were only slightly increased in ␥B(S11R/S11R) lenses. Calcium concentration was also increased in heterozygous ␥B(S11R/ϩ) mutant lenses (Fig. 5). This result confirmed that crystallin degradation likely resulted from an activation of calcium-dependent proteases in ␥B-S11R mutant lenses. Furthermore, the degree of calcium elevation and the amount of cleaved crystallins were correlated with the severity of the nuclear cataract in ␥B-S11R mutant lenses.

FIGURE 5. Total amounts of calcium, magnesium, sodium, and potassium in the lens measured by ICP-OES. Compared with WT lenses, ISCUSSION D calcium concentration was slightly increased in ␥B(S11R/ϩ) lenses; ␥ This work provides at least part of the molecular mechanism total calcium level was increased approximately fourfold in B(S11R/ ␥ S11R) lenses, whereas sodium, magnesium, and potassium levels were for how the B-S11R mutation leads to a nuclear cataract. only slightly increased. These differences were statistically significant Histologic and biochemical data indicate that the dense nuclear (n ϭ 3; P Ͻ 0.05). cataract caused by this mutation is related to the degeneration of inner fiber cells. Fiber cell degeneration is correlated with an increase of cleaved crystallin proteins and a disruption of actin to the cell boundary of inner mature fiber cells? Second, what filaments. Specific aggregation of ␥-crystallins in ␥B-S11R mu- leads to elevated calcium concentration? Third, how do actin tant lenses is likely to be one of the early events that trigger filaments undergo disassembly? One possible explanation is downstream changes, such as the loss of actin filaments and that ␥-crystallin aggregates disrupt or damage membrane-cy- the elevation of calcium concentration. Activation of calcium- toskeletal structures to increase the influx of extracellular cal- dependent proteases and disruption of cytoskeletal structures cium, and elevated intracellular calcium subsequently activates directly contribute to the degeneration of inner fiber cells, calcium-dependent proteases, such as calpains, leading to crys- which lead to severe nuclear cataracts. However, some impor- tallin degradation. tant questions remain to be answered. First, how do ␥B-S11R ␥-Crystallin mutations are among the most common causes mutant proteins cause the aggregation of ␥-crystallins adjacent of hereditary cataracts in humans and mice.5 Studies of several mutant ␥-crystallin proteins suggest that a decrease in the stability, solubility, or both of mutated proteins facilitates abnormal protein aggregates in the cytosol of fiber cells to cause distinct cataracts, such as “crystal-like” or lamellar cataracts.17,21–23 Others24 and we15 have also found that mutant ␥-crystallin proteins can form nuclear aggregates in differentiating fiber cells to inhibit the denucleation process, leading to severe cataracts with ruptured lenses in mice. The ␥D-P23T crystallin mutation leads to various types of cataracts in humans.25–27 Therefore, nonspecific protein aggregation of mutated ␥-crystallin proteins with reduced solubility or stability is not sufficient to explain how different types of cataracts can result from one particular mutation. Our experimental evi- dence suggests that mutated ␥-crystallin proteins may perturb other specific protein–protein interactions or important cellular events during fiber cell maturation that lead to distinct types of cataracts. Different types of cataracts often result from a combination of a specific gene mutation and other genetic modifier(s).15,28,29 The distribution of wild-type or mutant ␥B-crystallin proteins in the mouse lens has not been precisely determined. The ␥B-crystallin transcript is predominantly expressed in lens fibers, and the proportion of ␥B (and ␥C) proteins is reduced in the lens as mice age.30,31 The ␥B-I4F mutant protein is less heat FIGURE 4. Western blotting of lens crystallin proteins. Compared with ␣ wild-type (WT) lenses, cleaved ␣B- and ␥-crystallins were detected in stable in vitro, binds to -crystallin in lens homogenates, and 17 ␥ P7 mutant lenses (arrowheads). In P21 mutant lenses, cleaved forms of forms cytosolic aggregates in vivo. The B-I4F mutation re- all crystallins (␣A, ␣B, ␤, ␥) were detected (arrows). Total amount of sults in a lamellar cataract (from lens deep cortex to the ␤-actin remained unchanged between P7 and P21 wild-type and mu- nucleus), whereas the ␥B-S11R mutation causes an abnormal tant lenses. cortical ring and a dense nuclear cataract. Therefore, ␥B-crys-

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