aB-Crystallin in Lens Development and Muscle Integrity: A Gene Knockout Approach

aB-Crystallin in Lens Development and MuscleIntegrity: A Gene Knockout Approach

James P. Brady,1,2 Donita L. Garland,1 D. Earl Green,3,4 Ernst R. Tamm,5 Frank J. Giblin,6

and Eric F. Wawrousek1

PURPOSE. To study the role of aB-crystallin (aB) in the devel-oping lens and its importance in lens structure and function.

METHODS. Gene targeting in embryonic stem cells was used togenerate mouse lines in which the aB gene and its proteinproduct were absent. Gene structure and expression werecharacterized by genomic Southern blot, immunoblot, andNorthern blot analyses, and two-dimensional gel electrophore-sis. The gene knockout mice were screened for cataract withslit lamp biomicroscopy, and dissected lenses were examinedwith dark-field microscopy. Lenses and other tissues wereanalyzed by standard histology and immunohistochemistry.Chaperone activity was determined by heating lens homoge-nate supernatants and measuring absorbance changes.

RESULTS. In an unexpected result, lenses in the aB gene knock-out mice developed normally and were remarkably similar towild-type mouse lenses. All the other crystallins were present.The thermal stability of a lens homogenate supernatant wasmildly compromised, and when oxidatively stressed in vivowith hyperbaric oxygen, the knockout lenses reacted similarlyto wild type. In targeting the aB gene, the adjacent HSPB2gene, which is not expressed in the lens, was also disrupted.Loss of aB and/or HSPB2 function leads to degeneration ofsome skeletal muscles.

CONCLUSIONS. aB is not essential for normal development of atransparent lens in the mouse, and therefore is more dispens-able to the lens than the closely related aA-crystallin. It mayplay a small role in maintaining transparency throughout life.aB and/or the closely related HSPB2 is required to maintainmuscle cell integrity in some skeletal muscles. (Invest Ophthal-mol Vis Sci. 2001;42:2924–2934)

The a-crystallins are members of the small heat shock familyof proteins that, together with the b- and g-crystallins,

comprise the major water-soluble proteins of the vertebrateocular lens. The two a-crystallin proteins, aA- and aB-crystallin(hereafter known as aA and aB), are approximately 60% iden-tical with one another and share many common properties in

vitro, such as autokinase activity,1 interaction with cytoskeletalproteins,2–5 DNA binding,6 and the ability to act as molecularchaperones.7 However, there are differences between the twoproteins. Although both a-crystallins show sequence similarityto small heat shock proteins (sHSPs), only aB is inducible bystress.8,9 Also, aA is expressed at very high levels in the lensand is found in trace amounts in only a few nonlenticulartissues,10–12 whereas aB, although it is most highly expressedin the lens, is found at significant levels in a number of tis-sues.13–15 aB is particularly abundant in adult heart and skeletalmuscle, and its message has been detected very early in thedeveloping mouse heart and somites.16–17

The a-crystallins were originally thought to be lens-spe-cific structural proteins. However, the discoveries that aB isexpressed outside the lens and that it is stress inducible havebrought broader attention to the a-crystallins and to theirpotential usefulness for elucidating the functions and evo-lution of stress proteins. In addition, there is a growing listof human diseases in which aB has been shown to bemisexpressed, mislocalized, or otherwise involved.18 Onedisease, an autosomal dominant desmin-related myopathy,was recently shown to result from a missense mutation inthe human aB gene. This disorder is characterized by adult-onset muscular weakness, cardiomyopathy, and cata-racts.19,20 These clinical findings and the observation thataB is expressed very early in the developing heart andskeletal muscles suggest a critical physiological role for aB.

To elucidate the in vivo functions of a-crystallins, we gen-erated mice with targeted disruptions of the genes that encodethe aA and aB proteins. We previously reported that homozy-gous aA knockout mice have smaller lenses than those ofwild-type littermates and that progressive opacifications de-velop in the knockout lenses that apparently result, at least inpart, from the presence in lens fiber cells of inclusion bodiesthat contain high concentrations of aB.21 Herein, we presentthe initial characterization of mice with a targeted deletion thatdisrupts both the aB gene and the adjacent gene, HSPB2,which is an ancient duplication of aB.22 The existence of theHSPB2 gene, which lies approximately 1 kb upstream of aB,was reported after we produced the aB knockout mice. Similarto aB, HSPB2 is expressed in skeletal and cardiac muscleswhere it is often localized to the Z lines and has also beenshown to associate with and activate myotonic dystrophy pro-tein kinase.23 Unlike aB, HSPB2 is not expressed in the lens.22

Mice without both aB and HSPB2 are surprisingly viable andfertile and have no obvious perinatal defects, and their lensesremain as transparent as those of wild-type mice. However, asthey become older, aB/HSPB2 homozygous knockout miceshow postural defects and other health problems that appearto stem from progressive myopathy.

MATERIALS AND METHODS

Generation of aB/HSPB2 Gene Knockout MiceA l phage clone spanning approximately 16 kb of the aB/HSPB2 locuswas isolated from a 129Sv mouse genomic library (Stratagene, La Jolla,

From the 1National Eye Institute and the 3Pathology Section,Office of Research Services, National Institutes of Health, Bethesda,Maryland; the 5Anatomy Department, University of Erlangen-Nurnberg,Germany; and the 6Eye Research Institute, Oakland University, Roch-ester, Michigan.

Present affiliations: 2MetaMorphix, Inc., Baltimore, Maryland; 4USGeologic Survey, National Wildlife Health Center, Madison, Wisconsin.

Supported in part by National Institutes of Health Grant EY02027(FJG) and Core Center Grant EY05230.

Submitted for publication April 4, 2001; revised July 19, 2001;accepted August 6, 2001.

Commercial relationships policy: N.The publication costs of this article were defrayed in part by page

charge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Eric F. Wawrousek, NIH Bldg.6, Room 218,6 Center Drive MSC 2730, Bethesda, MD [email protected].

Investigative Ophthalmology & Visual Science, November 2001, Vol. 42, No. 122924 Copyright © Association for Research in Vision and Ophthalmology

CA). A targeting vector (Fig. 1A) was constructed by ligating tworestriction fragments: a 3-kb BamHI fragment, encompassing a portionof exon 3 and the 39 flanking sequences of the aB gene and a 5-kbNotI/NarI fragment encompassing a portion of exon 2 and the 39flanking sequences of the HSPB2 gene, into the vector pPNT24 onopposite sides of the phosphoglycerate kinase-neomycin phospho-transferase (PGK/neo) gene cassette (Fig. 1A). The targeting vectoralso included the PGK/herpes simplex virus thymidine kinase (PGK/HSVtk) gene cassette for negative selection. Electroporation of J1embryonic stem cells, selection and screening of targeted cells, andblastocyst microinjection were performed as previously described.21

Two independent embryonic stem (ES) cell clones were used to gen-erate two lines of knockout mice. DNAs from ES cells and knockoutmice were analyzed by PCR, using primers spanning, but not encodedwithin, the shorter arm of the knockout vector and by Southern blot,using the 3-kb BamHI fragment (Fig. 1A; triple line) as a probe. Oncestable lines of knockout mice were established, a simpler PCR protocolwas used for genotyping. PCR using three primers, 59-TAGCTTAATA-ATCTGGGCCA-39, 59-GGAGTTCCACAGGAAGTACC-39, and 59-TG-GAAGGATTGGAGCTACGG-39, in a 4:1:1 molar ratio produced a

310-bp product with the wild-type allele and a 600-bp product with theknockout allele. All work with mice conformed to the ARVO Statementfor the Use of Animals in Ophthalmic and Vision Research.

Western Blot Analysis of Lens andMuscle Proteins

Soluble and insoluble lens protein samples were prepared as previ-ously described.21 Mouse skeletal muscles were homogenized in TEbuffer (10 mM Tris; 1 mM EDTA) with 2.5% SDS, and protein concen-trations were determined using the bicinchoninic acid (BCA) assay(Pierce, Rockford, IL). Muscle proteins were separated on 12% SDS-polyacrylamide gels and then transferred to nitrocellulose, by a semi-dry transfer apparatus (Semi-Phor; Hoefer Scientific Instruments, SanFrancisco, CA). The blots were incubated with a 1:5000 dilution ofpolyclonal antiserum to recombinant human aB-crystallin (a gift fromJoseph Horwitz, Jules Stein Eye Institute, University of California at LosAngeles School of Medicine). Protocols for antibody incubation anddetection have been described.21

FIGURE 1. Targeted disruption ofthe aB and HSPB2 genes. (A) Struc-tures and relative orientation of theHSPB2 and aB genes in a wild-typemouse, gene-targeting construct, andstructure of the targeted locus. Dou-ble and triple lines beneath the wild-type locus indicate the 3-kb HpaI/XbaI and the 3-kb BamHI restrictionfragments used as hybridizationprobes for Northern and Southernblot analyses, respectively. (B) South-ern blot analysis of genomic DNAfrom wild-type (1/1), heterozygous(1/2), and homozygous (2/2)mice. Bands of the expected sizes forthe targeted allele were seen in the1/2 and 2/2 samples digested witheither HpaI (15 kb) or EcoRI (13 kb).(C) Immunoblot analysis of aB-crys-tallin in lens proteins from 1/1,1/2, and 2/2 mice showed that aBis absent in 2/2, and its levels weredecreased in 1/2 mice. A minorcross-reactivity of this antibody to-ward a slightly smaller protein wasobserved in the knockout lens. (D)Northern blot analysis of lens andmuscle tissues from wild-type (1/1)and homozygous knockout (2/2)mice, by using a probe specific forHSPB2 (double line in A). HSPB2mRNA was not detected in any tis-sues from knockout mice, nor in thelens of wild-type mice. The blot wasreprobed with a b-actin probe thatcross-hybridizes with a-actin in mus-cle tissues (D, bottom). N, NarI; B,BamHI; E, EcoRI; H, HpaI; H, heart;G, gastrocnemius muscle; L, lens; S,soleus muscle; P, plantaris muscle.

IOVS, November 2001, Vol. 42, No. 12 aB-Crystallin Knockout Mice 2925

Northern Blot to Detect HSPB2 mRNA

Northern blot analysis was performed using standard protocols.25 RNAwas extracted from lens, heart, soleus, plantaris, and gastrocnemiusmuscles of 16- to 17-week-old knockout and wild-type mice. Total RNA(10 mg for lens, heart and gastrocnemius; 5 mg for soleus and plantaris)was used, and before blots were formed, a gel lane containing RNA sizemarkers (Life Technologies, Rockville, MD) was removed and stained.The blot was probed with a 3-kb HpaI/XbaI genomic fragment (Fig.1A; double line), spanning the entire HSPB2 gene and approximately0.8 kb of its 39 flanking sequence, and then reprobed with a b-actinprobe (Oncor, Rockville, MD).

Slit Lamp Biomicroscopy

Mouse eyes were dilated with eye drops containing 1% tropicamidefollowed several minutes later by eye drops containing 2.5% phenyl-ephrine hydrochloride. Approximately 15 minutes later, the mice wereanesthetized with an intraperitoneal injection of 0.015 to 0.017 ml2.5% 2,2,2-tribromoethanol per gram body weight and immediatelyexamined with a slit lamp. The slit illumination was always at a 45°angle to the observation optics.

Histology

Eyes were removed, gently pierced in the limbal region, and fixed in4% buffered glutaraldehyde for 20 minutes, followed by 4% bufferedformaldehyde or 4% buffered formaldehyde alone. For a milder treat-ment, eyes were occasionally fixed in 4% buffered paraformaldehydefor 25 minutes, rinsed for 30 minutes in PBS, and placed in 80% ethanolfor several days. They were embedded in methacrylate or paraffin,sectioned, and stained with hematoxylin and eosin (H&E). Hindlimbsand spine regions were fixed in 4% buffered formaldehyde, decalcified,embedded in methacrylate or paraffin, sectioned, and stained withH&E. Tongue was similarly processed, but the decalcification step wasomitted.

Protection from Heat-Induced Denaturation Assay

Pairs of lenses from wild-type, heterozygous and homozygous aBknockout, and homozygous aA knockout mice were disrupted in 1 mlof buffer (0.1 M sodium phosphate [pH 7.4]; 0.1 M sodium chloride).The homogenate was centrifuged for 10 minutes at 4°C at 14,000g ina microfuge. The supernatant was removed and the pellet was reho-mogenized with another 1 ml buffer. After centrifugation, each super-natant was pooled with the first. Protein concentrations were deter-mined by BCA assay (Pierce) and samples were diluted to 3.1 mg/mlprotein. Samples were placed in a temperature-controlled (54°C or63°C) spectrophotometer cuvette holder at time 0, and absorbance at360 nm was monitored.

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis and image analysis were per-formed, essentially as previously described.26 Briefly, lenses frozen ondry ice immediately after dissection and maintained at 270°C werethawed in a protease inhibitor mix (P8340; Sigma, St. Louis, MO) thenhomogenized in 9 M urea, 2% NP40, 10 mM dithiothreitol (DTT), 2%ampholytes (Resolyte 3.5-10), and centrifuged at 16,000g for 20 min-utes. The supernatant was run in the first-dimension isoelectric focus-ing strip, using dry-strip gels (pH 3–10 nonlinear; Immobiline DryStrip;Amersham Pharmacia Biotech, Piscataway, NJ), and in the seconddimension, using 15% to 18%.SDS polyacrylamide gradient gel. Gelswere stained with Coomassie blue, cleared with water, scanned on apersonal densitometer (Molecular Dynamics, Sunnyvale, CA), and an-alyzed by computer (Phoretix, ver. 5.0; Phoretix International, New-castle-upon-Tyne, UK) software.

Immunohistochemistry

Tissues used for immunohistochemistry were fixed in 4% bufferedparaformaldehyde. Spine and hindlimb tissues were then decalcified.All tissue preparations were embedded in paraffin and sectioned ontosilane-coated slides. Immunohistochemistry was performed using im-munoperoxidase reagents in avidin-biotin complex (ABC) and sub-strate kits (Vectastain Universal Elite and VIP, respectively; VectorLaboratories, Burlingame, CA), according to the manufacturer’s in-structions. Serial sections were immunostained, and, because mousemonoclonal antibodies were being used with mouse tissues, one slidein each experiment was run without any primary antibody, as a controlfor nonspecific detection by the anti-mouse IgG secondary antibody.One slide was also stained with H&E. Monoclonal antibodies againstmyosin heavy chains, (MHC fast and MHC slow; Sigma) were used at1:500 dilution, and the monoclonal antibody against desmin (Dako,Carpinteria, CA) was used at dilutions of 1:20 to 1:75.

Transmission Electron Microscopy

Tongues were sliced with a razor blade into approximately 3-mmcross-sections to aid penetration of fixative. The tongue sections andsoleus and extensor digitorum longus (EDL) muscles dissected fromhindlimbs of knockout (53-week-old) and wild-type (57-week-old)mice, were fixed in freshly prepared 2.5% glutaraldehyde and 2.5%paraformaldehyde in 80 mM cacodylate buffer (pH 7.2). After fixation,the tissues were equilibrated in cacodylate buffer for at least 24 hoursand then postfixed with 2% OsO4 and embedded in Epon (Roth,Karlsruhe, Germany). Ultrathin sections were contrasted with leadcitrate and uranyl acetate and examined with a transmission electronmicroscope (EM 902; Carl Zeiss, Oberkochen, Germany).

RESULTS

Generation of aB/HSPB2Knockout Mice

Figure 1A shows the strategy that was used to knock out the aBgene and concomitantly the adjacent HSPB2 gene. All theprotein-coding sequences through the middle of the third andfinal exon of aB and 1.6 kb of DNA encompassing the putativetranscriptional regulatory sequences for aB and HSPB2 as wellas most of the HSPB2 protein-coding sequences were elimi-nated in the targeted chromosome. Genomic Southern blotanalysis demonstrated the expected rearrangements of the aBand HSPB2 genes in heterozygous and homozygous knockoutmice (Fig. 1B). Deletion of an EcoRI site in the targeted alleleresulted in an increase from 9 to 13 kb of the probe-containingEcoRI fragment, and replacement of the sHSP genes with theshorter PGK/neo cassette decreased the size of the probe-containing HpaI fragment from 18 to 15 kb. Western blotanalysis of soluble and insoluble proteins from the lens, thetissue with the highest expression level of aB, confirmed thatthe full-length form and two C-terminally truncated forms of aBwere all decreased in heterozygous and absent in homozygousknockout mice (Fig. 1C). Further evidence for the absence ofaB is the absence of an aB signal in immunoblots of skeletalmuscle and heart (not shown) and the loss of immunologictolerance to aB as a self-antigen in knockout mice (Igal Gery,personal communication, November 1996).

The closely linked HSPB2 gene was discovered subsequentto the production of these knockout mice, and because avail-able sequence data22 suggested we had disrupted this genealso, Northern blot analysis was performed to confirm this.Figure 1D shows that HSPB2 mRNA, which was previouslydetected in adult heart and skeletal muscles but not in lenses ofwild-type rats,22 was absent from heart and skeletal muscles ofhomozygous aB/HSPB2 knockout mice, but was present in

2926 Brady et al. IOVS, November 2001, Vol. 42, No. 12

wild-type mouse muscle tissues, confirming that the HSPB2gene, in addition to the aB gene, was functionally inactivated.Figure 1D also confirms that HSPB2 is not expressed in thelenses of normal mice.

aB/HSPB22/2 mice are born at nearly normal Mendelianratios from matings between heterozygous (1/2) animals(20% of 200 such offspring were 2/2). At birth, the knockoutmice exhibit no obvious mutant phenotypes.

Characterization of Lens Structureand Development

Analysis of lenses from 11-week-old mice revealed average lensweights of 5.5 6 0.2, 5.3 6 0.2, and 5.1 6 0.2 mg for wild-type,heterozygous, and homozygous aB-crystallin/HSPB2 knockoutmice, respectively. Measurement of the equatorial and axialdimensions of whole eyes and lenses from the same micerevealed no significant differences (not shown). This is in starkcontrast to aA knockout mice in which the lens weight isreduced by approximately 30% and the axial and equatorialdimensions are decreased by approximately 15%.21

The slit lamp micrographs (Figs. 2A, 2B) show little if anydifference in light scattering between the wild-type and knock-out lenses. There is a basal level of light scattering in the lensesof 129Sv mice that manifested itself in slit lamp examination asan apparent opacity at the edge of the lens opposite the slitillumination. This may have been due to the recently discov-ered absence of the lens-specific beaded filament cytoskeletalproteins, cp49 and filensin, in lenses of 129S strains of mice.27

Age-matched knockout mice, particularly at older ages, appearto have slightly more of this light scattering. Examination ofdissected lenses with dark-field illumination (Figs. 2C, 2D)confirms the similarly low light scattering in wild-type and aBknockout lenses, which contrasts with the high degree of lightscattering in the aA knockout lenses. Histologic examinationof lenses revealed no obvious differences between wild-typeand aB knockout specimens (Fig. 3). In contrast, mice inwhich the closely related aA gene is disrupted (Figs. 3E, 3F)show development of lens opacities before 7 weeks of age, andhistologic abnormalities even earlier,21 which progress in se-verity with age (Figs. 2A, 2C, 2D).

Certain anomalies were observed in both wild-type and aBknockout lenses, depending on the method of fixation used.Fixation with 4% buffered formaldehyde alone (Figs. 3G–J)caused an invagination of the lens at the equator that wassimilar in wild-type (Fig. 3G) and aB knockout (Fig. 3I) mice.When eyes were fixed with 4% buffered glutaraldehyde for 20minutes followed by 4% buffered formaldehyde, small vacuoleswere observed in the outer cortex of lenses from both wild-type (Figs. 3K, 3L) and aB knockout (Figs. 3M–P) mice. Sam-ples fixed with buffered formaldehyde alone (Figs. 3G–J) orformaldehyde for 25 minutes followed by 80% ethanol (notshown) did not exhibit these vacuoles. Because these vacuolesappeared with only one method of tissue fixation and indepen-dently of the presence or absence of aB, they may have beenartifactual. Cytoplasmic inclusion bodies present in the lensnuclei of aA knockout mice (Fig. 3F) were not observed in thelenses of aB knockout mice (Fig. 3D). These results clearlydemonstrate that the lens can much more easily accommodatethe loss of aB function than it can the loss of aA function.Because the HSPB2 gene is not expressed in the lens22 (Fig.1D), its disruption is not expected to affect the lens.

Crystallin Expression in Knockout Lenses

Because the lenses of aB knockout mice appear normal andtransparent, we suspected that the distribution of other major

crystallins was not significantly affected by the absence of aB.Two-dimensional gel electrophoresis of lens proteins (Fig. 4)followed by image analysis, which detects spots too faint to beseen by the naked eye, revealed that all the major and minorcrystallin spots present in wild-type mice, except, of course,for aB and modified forms of aB, were also present in theknockout mice. There were no new or additional crystallinspots detected in the knockout mice. aB, its mono- and di-phosphorylated forms, and the phosphorylated and unphos-phorylated forms of the aB C-terminal truncation (amino acids171 to 175 removed) are all absent in the aB knockout lens.

FIGURE 2. Examination of wild-type and aB knockout lenses. (A) Slitlamp examination of eyes of 17-week-old wild-type and aB knockoutmice and an 18-week-old aA knockout mouse. The aA knockout lensis completely opaque, whereas the wild-type and aB knockout lensesremain transparent. (B) Slit lamp examination of eyes of 36-week-oldwild-type and aB knockout mice. The aB knockout lenses scatteredlight slightly more than the basal level of the wild-type 129Sv strain. (C)Dark-field examination of lenses from 26-week-old wild-type, aBknockout and aA knockout mice (top), and a 19-week-old aA knockoutmouse (bottom). (D) Dark-field examination of lenses from 39-week-old wild-type and aB knockout mice, and a 19-week-old aA knockoutmouse. The wild-type and aB knockout lenses appear similarly trans-parent, whereas the aA knockout lenses are opaque. The zonulesremain attached to the lens capsule and are visible at the equators of allthe lenses. The dark-field illuminator had four light sources that arereflected by the edges of each lens. Wt, wild-type; aB ko, aB knockout;aA ko, aA knockout.


Protection against Stress in the Lens

Because aB is an sHSP and molecular chaperone, we investi-gated whether lenses without aB were more susceptible todamage. The ability of lens proteins to resist heat-induceddenaturation was studied (Fig. 5). When the soluble fraction oflens homogenate from wild-type mice was heated to 54°C,there was little increase in turbidity of the solution over the60-minute experiment, indicating that most proteins remainedsoluble and that these lenses have thermal protective capacity.The lens homogenate supernatant without aB showed a slowincrease in turbidity (and therefore, protein denaturation) be-ginning at 30 minutes and reached twice the level in wild-typelenses at 60 minutes, suggesting that the overall protectiveactivity in the aB knockout lens was mildly impaired (Fig. 5A).When the lens homogenate supernatants were heated to 63°C,the difference between wild-type and aB lenses became morepronounced, with turbidity of the knockout homogenate su-pernatant reaching three times that of wild type at 60 minutes(Fig. 5B). There was still a high degree of thermal protection in

the aB knockout lenses compared with aA knockout lenses,which exhibited almost no protective capacity, with much ofthe protein precipitating after 15 minutes (Fig. 5B).

We also tested in vivo, the ability of aB knockout lenses toresist oxidative stress. Exposure of humans and animals tohyperbaric oxygen has been shown to induce an increasedlevel of lens nuclear light scattering or nuclear cataract.28–31

Repeated exposure of mice to hyperbaric oxygen (2.5–3.2atmospheres of O2 for 2.5–3 hours per exposure, three expo-sures per week) over a period of 5.5 months induced increasedlens nuclear light scattering equally well in wild-type andknockout mice (not shown), suggesting that in vivo, aB doesnot play a major role in protecting the lens against oxidativedamage. This is in agreement with the results of Kannan etal.,32 who demonstrated that the level of reduced glutathione(GSH) in lenses without aB is equal to that in wild-type lenses,whereas lenses without aA exhibit a severe decrease in GSHlevel, and with the results of Andley et al.,33 who demonstratedthat aB is significantly less effective than aA in protecting lenscells against stress. Development of lens opacity in aA knock-out mice at an early age, even in the absence of added oxidativestress, precludes similar hyperbaric oxygen experiments fromproviding useful data on aA knockout mice.

FIGURE 4. Two-dimensional gel electrophoresis analysis of proteins inaB knockout lenses. Proteins from 10-week-old wild-type (A) and11-week-old aB knockout (B) mice were analyzed. (A) Wild-type pro-file shows aB spot (large arrowhead) and several aB phosphorylationproducts and C-terminal truncations (arrows). (B) The main aB spotand all the minor spots corresponding to modified aB are absent in theaB knockout lens; vacant positions are indicated by arrowhead andarrows as in (A). All other major and minor spots, including those notvisible to the naked eye but detected by the computer software, arepresent in both wild-type and aB knockout lenses.

FIGURE 3. Histologic examination of lenses from wild-type and knock-out mice. H&E-stained sections of lenses from (A) 7.5-week-old wild-type, (C) 9-week-old aB knockout, and (E) 8-week-old aA knockoutmice. (B, D, F) Higher magnification of lens regions delimited byrectangles in (A), (C), and (E), respectively. aB knockout lenses,similar to wild-type lenses, did not have the inclusion bodies that wereevident throughout the nuclear and inner cortical fiber cells of aAknockout lenses. (G) Bow region and (H) epithelial region of a 10-month-old wild-type mouse lens that had been fixed in 4% formalde-hyde. (I) Bow region and (J) epithelial region of a 10-month-old aBknockout mouse lens that had been fixed in 4% formaldehyde. Thewild-type and aB knockout lenses were similar, with an artifactualinvagination at the equator. (K) Bow region and (L) epithelial region ofa 10-month-old wild-type lens that had been fixed in glutaraldehydefollowed by formaldehyde. (M, O) Bow regions and (N, P) epithelialregions of 10-month-old aB knockout mouse lenses that had been fixedin glutaraldehyde followed by formaldehyde. The lenses were similarwith vacuoles in the cortex of both wild-type and aB knockout. wt,wild-type; ko, knockout. Scale bar, (G–P) 100 mm.


Health Problems in Aging Knockout Mice

The growth curves of male and female knockout mice paral-leled those of wild-type 129Sv until approximately 40 weeks ofage (data not shown); but thereafter, the knockout mice con-sistently lost weight and eventually lost most of their body fat.The mice also displayed a hunched posture resulting fromdevelopment of a severe curvature of the spine (kyphosis) thatcould be readily observed in radiograms of 12-month-old mice(Fig. 6). Histologic examination of the hunched mice revealeddegenerative osteoarthritis of the intervertebral facet joints.Although not uncommon in much older wild-type mice, thisprocess appears to be greatly accelerated in the knockoutmice. Because both aB and HSPB2 are expressed in musclecells, we suspected that the underlying cause of the observedkyphosis was related to altered function of muscles associatedwith the axial skeleton. In histologic examination of 65-week-old knockout mice exhibiting the hunched posture, the heartappeared normal (not shown), but severe degeneration ofsome skeletal muscles was readily apparent (Fig. 7) with themost severely affected muscles being in the posterior tongue(Figs. 7B, 7D), head, and surrounding the axial skeleton (Figs.7F,7 H). Limb muscles were affected to a lesser extent (notshown). None of these degenerative changes was observed inthe skeletal muscles of age-matched heterozygous (not shown)or wild-type mice (Figs. 7A, 7C, 7E, 7G).

In an area of the posterior tongue of a 65-week-old knock-out mouse, degenerated muscle was nearly completely re-placed by fatty tissue (Figs. 7B, 7D). The few muscle cellsremaining were quite abnormal and probably would have dis-appeared soon thereafter. Axial skeletal muscles exhibited a

variety of alterations that may represent a progression of thedegenerative processes. These include migration of nuclei intothe sarcoplasm, hyalin degeneration of the sarcoplasm, vacu-olization of the sarcoplasm, infiltration of macrophages, fibro-sis, and fatty replacement of muscle cells. Several of theseabnormalities can be seen in Figures 7D and 7H. Muscle weak-ness in the tongue and head regions probably leads to animpaired ability to obtain nourishment, accounting for the lossof weight and body fat observed in older mice; and decreasedaxial skeletal muscle function probably leads to the accelerateddegenerative vertebral osteoarthritis and hunched posture ob-served in older knockout mice.

Because the muscle degeneration in older mice was severeand apparently the consequence of chronic muscular dystro-phy, we examined knockout mice at several younger ages (notshown). At 7 weeks of age muscles appeared relatively normal,exhibiting only very minimal axial muscular dystrophy. By 20weeks of age, the axial muscular dystrophy had progressed inseverity, exhibiting some occasional fibrosis, fatty infiltration,and myositis, suggesting the chronic nature of this condition.By this age, muscles of the head were also affected, and thediaphragm and select hindlimb muscles (e.g., the soleus) weremildly affected. At 40 weeks of age, the accumulated damageto the axial muscles was more severe, with fibrosis and fattyinfiltrates more pronounced. Muscles of the head, particularlythose of the hyoid apparatus, showed marked degeneration.The soleus muscle in the hindlimb was moderately affected. Atthis age the mice began to lose body mass and exhibit ahunched posture. At all ages examined, the most severelyaffected muscle cells were generally located adjacent to boneor at tendinous insertions and occasionally deep within musclebundles.

Ultrastructure of the Degenerating Muscle Cells

Electron microscopy revealed degenerative changes at the ul-trastructural level (Fig. 8). The appearance of amorphous,flocculent, electron-opaque material (FEOM) interspersed be-tween myofibrils was seen in mildly affected cells (Fig. 8,arrows), probably signifying early events in the degenerativeprocess. A mildly affected tongue cell (Fig. 8A) with only asmall area of FEOM was adjacent to cells with relatively normalappearances. As the degenerative process progressed in both

FIGURE 6. Radiographs of an 12-month-old wild-type mouse (top) anda 10-month-old knockout mouse (bottom) clearly illustrate the severeanterior kyphosis and emaciation in aging knockout mice.

FIGURE 5. Protection against heat-induced denaturation of proteins inknockout lenses. The soluble fractions of lens homogenates, adjustedto 3.1 mg protein/ml, were heated to 54°C (A) or 63°C (B) in spec-trophotometer cuvettes and absorbance at 360 nm monitored. aBknockout lenses (aB2/2) had less thermal protective capacity thanwild-type (wt), but much more than aA knockout lenses (aA2/2).


tongue (Figs. 8A, 8C, 8D) and soleus (Fig. 8E), the relativeproportion of FEOM increased within the cell, with a concom-itant loss of myofibrils (black arrowheads) until there werefew, if any, myofibrils remaining (Figs. 8C, 8E). Consistent withhistologic findings (Fig. 7), large vacuoles (identified by aster-isks) were present in the later stages of muscle cell degenera-tion in both tongue (Fig. 8D) and soleus (Fig. 8E). Degenerativechanges were not observed in EDL muscles from the sameknockout mice (not shown). None of the degenerative changesobserved in muscles of knockout mice was seen in the corre-sponding tissues of age-matched wild-type mice (Fig. 8B).

DISCUSSION

aA- and aB-crystallins, members of the family of sHSPs, areboth expressed at high levels in the vertebrate lens. WhereasaA is expressed at very low levels in a few nonlenticulartissues,11,34 aB is expressed at significant levels in many othertissues13,14 and, as the family name implies, is induced by heatand other stresses.8,9,18 A point mutation in the aA gene hasbeen linked to autosomal dominant cataract in humans,35 andsimilarly, a point mutation in the aB gene has been linked to adesmin-related myopathy with associated cataract.20

Although aA and aB share a high degree of homology andform heteromultimeric aggregates,36,37 there appear to be sig-nificant differences between these proteins. In vitro, aB hasbeen shown to be less thermodynamically stable,38 more sus-ceptible to heat-induced conformational changes,39 a less ef-fective chaperone,40,41 and more preferentially dissociated

from native a-crystallin aggregates by chaotropic agents42 thanaA. Unlike aA, aB does not exhibit markedly increased autoki-nase activity when dissociated to the tetrameric state.43 In cellculture, aB has been shown to be less effective than aA inpreventing lens epithelial cells from ultraviolet light or stauro-sporine-induced apoptosis,33 and addition of aB, but not aA, tocultured lens cells has been shown to induce morphologicchanges reminiscent of differentiating fiber cells.44

We have studied the roles of the two a-crystallins in vivo byremoving them by gene targeting in the mouse. Previously, weshowed that the absence of aA results in microphthalmia,decreased lens size and weight, cataract formation before theage of 7 weeks, formation of aB-containing cytoplasmic inclu-sion bodies in lens fiber cells by 4 weeks of age, and a majorshift of lenticular aB from the soluble to the insoluble phase.21

It is therefore possible that a primary function of aA is main-taining the solubility of high concentrations of aB in lens cells.In the present study, we deleted the aB and the closely relatedHSPB2 genes from the mouse. In contrast to the lens devasta-tion observed in the aA knockout mouse, the lenses of the aBknockout mice were remarkably similar to those of wild-typemice and remained so throughout life, suggesting that aA ismore important in lens development and maintenance of lenstransparency than aB.

The sizes of aB knockout lenses were the same as those ofwild type, and the lens mass of knockout mice was onlyapproximately 7% less than wild type, compared with theapproximately 30% mass reduction and 15% reduction axialand equatorial dimensions of the aA knockout lenses. It is

FIGURE 7. Histologic examinationof tongue and spinal muscles fromwild-type and knockout mice. Sec-tions through the posterior tongue ofa (A) 68-week-old wild-type mouseand (B) a 65-week-old knockoutmouse reveal an area in which mus-cle was almost completely replacedwith fatty tissue (white area in thecenter of the tongue). (C) A magni-fied view of the boxed area in (A)shows healthy muscle cells witheven staining and peripheral nuclei.(D) A magnified view of the boxedarea in (B) shows remnants of only afew muscle cells that are unevenlystained (blue arrow) or vacuolated(black arrows) amid a field of fattytissue. Cross section of the spine andsurrounding tissue of (E) a wild-typemouse and (F) an aB knockoutmouse. (G) A magnified view of theboxed area in (E) shows healthymuscle cells of uniform size, evenlystained, with nuclei at their periph-eries. (H) A magnified view of theboxed area in (F) shows irregularlysized muscle cells exhibiting varyingdegrees of degeneration. These in-clude cells with centralized nuclei(black arrowheads) indicative ofmuscle degeneration and regenera-tion; cells stained unevenly (blue ar-rows), with normal staining at theperiphery and dark staining at thecenter, suggesting hyaline degenera-tion of the sarcoplasm; vacuolatedcells (black arrows) signaling de-

struction of the muscle cell; and fatty infiltrate (blue arrowheads), indicating loss of dead muscle cells. Representative, not all, cells of each typeare indicated. It is interesting to note that the affected muscles are adjacent to relatively normal-appearing muscles (✽ in H). SC, spinal cord.


unclear whether this slight mass reduction is due to decreasedcell number or a decreased average mass per cell in the knock-out lenses. Despite the small difference in mass, the lenses ofaB knockout and wild-type mice appeared very similar in grossmorphology and histologically, and although slit lamp exami-nation of the aB knockout lenses often produced slightly morebasal light scattering than did wild-type lenses, aB knockout

and wild-type lenses dissected and directly compared in dark-field illumination exhibited a similar amount of light scattering.Such discrepancies between slit lamp examinations and exam-inations of dissected lenses have been previously documentedin mice.45 In both slit lamp and dark field, light scattering wasfar less in lenses of aB knockout mice than those of aAknockout mice.

It has recently been reported that lenses from 129Sv micedo not have the lens-specific beaded filament cytoskeletal pro-teins cp49 and filensin, as a result of a mutation in the cp49gene.27 This deficit may be responsible, at least in part, for thebasal level of light scattering seen in the lenses of both wild-type and aB knockout mice that are in the mouse 129Svbackground. The absence of this cytoskeletal component mayalso contribute to the vacuoles observed in the outer cortex oflenses fixed with glutaraldehyde followed by formaldehyde(Figs. 3K–P), regardless of whether these vacuoles are truereflections of the in vivo condition or artifacts of tissue prep-aration resulting from a weakened cytoskeleton.

A key feature of the cataract in aA knockout mice is thepresence of cytoplasmic inclusion bodies containing mainlyaB,21 indicating that the presence of aA is essential to main-taining the solubility of aB in lens nuclear and inner corticalfiber cells. In the absence of aB, we saw no indication ofinclusion bodies containing aA, suggesting that aA is morestable in the lens milieu than aB and does not require itsaggregation partner, aB, to remain in solution. This apparentpropensity of aB to form inclusion bodies at high concentra-tion is of particular interest, because aB has been found to bea constituent of inclusion bodies in a variety of pathologicconditions46 in which aB is upregulated, including Rosenthalfibers in Alexander disease,47 cortical Lewy bodies in Lewybody disease,48 Mallory bodies in alcoholic liver disease,48 andAlzheimer disease plaques and neurofibrillary tangles.49–51

The overall chaperone activity in the aB knockout lens wasonly mildly diminished compared with that of the aA knockoutlens, as demonstrated by protection against heat-induced ag-gregation of proteins in a lens homogenate supernatant. Thethermal protection assay has recently been shown to giveresults comparable to other types of chaperone assays.41 Con-tributing to the severely decreased chaperone activity in aAknockout lenses, compared with aB knockout lenses, may bethe weaker chaperone activity of aB compared with that ofaA41 and the fact that in aA knockout lenses not only is the aAabsent, but a sizable portion of the aB is in the insoluble phaseand therefore is also absent from the lens homogenate super-natant used for the assay.21 Hyperbaric oxygen treatment of

Š

FIGURE 8. Electron micrographs of tongue and soleus muscle fromknockout mice (A, C, D, E) showing various degrees of muscle celldegeneration, and from wild-type mice (B). In one muscle cell (A, top)in the tongue of a knockout mouse, an amorphous, FEOM (A, C;arrows) is interspersed between regularly shaped myofibrils, whereasan adjacent cell (A, bottom) exhibits no such abnormalities. This areaof the lower muscle cell is a mitochondria-rich region (mitochondriaare dark ovoid bodies). (B) Normal muscle cell architecture is seen ina wild-type mouse soleus muscle. The regular, repeating sarcomericstructure, punctuated by the dark Z line, is readily evident; nothingresembling FEOM was observed in wild-type muscle. (C) In a severelydegenerated knockout tongue muscle cell, an isolated, relatively nor-mal-looking myofibril bundle (arrowheads) is surrounded by theFEOM (arrow). (D) In this cross section through a severely affectedknockout tongue cell, two vacuoles with irregular edges are present(✽). (E) Cell from the hindlimb soleus muscle exhibits a similar patternof FEOM. At this advanced stage of degeneration, an isolated myofibrilbundle (arrowheads) is surrounded by the FEOM, and a large vacuoleis evident (✽).


mice, used to oxidatively stress lenses in vivo, showed nodifference in loss of lens transparency between aB knockoutand wild-type mice. This is consistent with the relatively nor-mal chaperone activity in the aB knockout lenses and therelatively normal levels of GSH in lenses of aB knockout miceat all ages.32

Although the lens seems unaffected by the absence of aBand HSPB2, this mutation causes a severe phenotype in oldermice that is characterized by hunched posture, loss of bodymass after 40 weeks of age, and severe muscle cell degenera-tion, but in only some muscles. Because both aB and HSPB2are expressed in skeletal muscle23,52 and because both geneswere disrupted in this knockout mouse, we cannot ascribe thisphenotype specifically to either of the proteins; therefore, themuscle phenotype observed may result from the absence ofeither or both of these closely related sHSPs.

Expression of aB in skeletal muscle is highest in the mostoxidative, slow-twitch type I muscle cells, intermediate infast-twitch type IIA cells, and lowest in the glycolytic, fast-twitch type IIB muscle cells,15,53 consistent with its role as astress protein, and has been shown to colocalize with desminat the Z bands.54 The presence of muscle cell degeneration inthe cell type I–containing soleus but not in the type II plantarisor EDL suggests, but does not prove, that the oxidative type Imuscle cells, which normally would contain the highest levelsof aB, are the degenerating cells. Attempts to identify thedegenerating muscle cell type by immunostaining with anti-bodies for fast- or slow-twitch myosin heavy chains were un-successful, with the degenerating cells staining for neither type(data not shown). This could have been due to loss of themyosin heavy chain antigen in the severely degenerated mus-cle cells. Indeed, in a French family with desmin-related my-opathy caused by a point mutation in aB,20 the muscles con-tained cells with a “rubbed-out” appearance when stained foreither myosin adenosine triphosphatase (ATPase) activity oroxidative activity, suggesting the loss of myosin in thesecells.19 Vacuoles with irregular edges were also observed.These anomalies were found exclusively in type I fibers.19

In the French desmin-related myopathy,19,20,55 a mutant aBprotein is present that becomes insoluble and forms denseelectron-opaque bodies in affected muscle cells.20 The R120Gmutant form of aB in this family was shown in vitro to adoptan irregular structure, have drastically reduced chaperonefunction, and be found in the insoluble material after chaper-one assays.56 This is quite different from the situation in our aBknockout mice, in which the aB was simply absent. In thiscase, there was no mutant protein with abnormal activity tointerfere with cellular processes; thus, we were looking purelyat the effect of deletion of the normal protein(s) from the cell.In this regard, some of the anomalies found in our knockoutmice are similar to those found in the French family: emacia-tion, difficulty swallowing, abnormal gait, weakness of musclesin the neck and trunk,19,20 and increased immunohistochemi-cal staining for desmin in severely affected muscle cells (notshown), suggesting that the loss of normal aB function maycontribute to both phenotypes.

As stated earlier, we cannot rule out the possibility that theother deleted gene, HSPB2, may be responsible for the ob-served muscle phenotype. HSPB2, also known as myotonicdystrophy protein kinase binding protein (MKBP), has beenshown to activate myotonic dystrophy protein kinase (DMPK)and protect it from heat-induced inactivation23 and is upregu-lated in myotonic dystrophy. Similar to our knockout mice,mice without DMPK show age-related changes in head andneck muscle fibers57 and late-onset, progressive skeletal myop-athy, with elevated muscle fiber degeneration and fibrosis.58 Ittherefore seems plausible that the absence of HSPB2/MKBP

contributes to the observed phenotype by failing to activateDMPK.

Similar to aB, HSPB2 has been localized to the Z lines inskeletal muscle, suggesting that in addition to its role of acti-vating DMPK, it may also play a more direct role in stabilizingthe sarcomeric structure.23 However, HSPB2, with its aggrega-tion partner HSPB3, has been shown in muscle cells to formoligomeric complexes that are separate from the complexesformed by aB, HSP25, and HSP20. Thus, although they colo-calize at Z lines, the two types of sHSP complexes may havedifferent protective functions there52 and may both be essen-tial for maintenance of sarcomeric structure in oxidative mus-cle cells.

Because aB is expressed at high levels very early in embry-onic heart development, and both aB and HSPB2 are expressedin the fully formed heart, we originally suspected that the geneknockout would cause cardiac problems resulting in embry-onic or neonatal lethality. We were surprised to observe thatthe hearts of knockout mice appeared histologically normal,even at older ages, and functioned sufficiently in mice living ina controlled animal facility environment. However, furtherstudies of cardiac function under a variety of conditions arebeing conducted by collaborators with cardiology expertise.

In summary, we have shown that aB-crystallin and HSPB2are not essential for viability or reproduction of the laboratorymouse, aB is not essential for proper lens development, and aBand/or HSPB2 is essential to maintaining integrity of someskeletal muscles. Our data suggesting that aB contributes sig-nificantly less to lens development and function than aA agreewith recent evidence that aA is a better chaperone than aB,41

that aA can protect lens cells better than aB,33 and that loss ofaA leads to a severe decrease in the reducing capacity withinthe lens, as measured by GSH level, whereas loss of aB has noeffect on GSH level.32 This finding also supports the notion thatafter the ancient gene duplication event that gave rise to thetwo a-crystallin genes, aA evolved to perform critical functionsin the lens whereas aB, still highly expressed in the lens,evolved into a more general stress protein. Therefore, althoughaB may attract broader interest because of its functions inmany extraocular tissues such as muscle, aA appears to be themore important protein in lens development and function.

Acknowledgments

The authors thank Mahesh Mankani (National Institute of Dental Re-search, Bethesda, MD) for performing x-ray analysis of the aB knockoutmice; Victor Leverenz and students, Navneet Brar, Valery Heller, Whit-ney Lakin, Allan Rinke, and Zhilin Yan (Oakland University), for assis-tance in the treatment of the mice with hyperbaric oxygen; YvonneDuglas-Tabor (National Eye Institute) for assistance with two-dimen-sional gel electrophoresis; and Steven Lee (National Eye Institute) forassistance with mouse genotyping.

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aB-Crystallin in Lens Development and Muscle Integrity: A Gene Knockout Approach

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