Lack of dystrophin protein Dp71 results in progressive cataract formation due to loss of fiber cell organization

Animals

Dp71 knockout (KO-Dp71) mice and their littermates were bred in our laboratory. Mice were originally generated following the method described [14] and then bred in our laboratory. Briefly, the first and unique exons of Dp71 and a small part of the Dp71 first intron were replaced by a sequence encoding a β-gal-neomycin-resistance chimeric protein (b-geo), only abolishing the expression of Dp71.

Mice were identified through an analysis of PCR products using the following oligonucleotide primers: Dp71F, 5’-ATG GAA CAG CTC AAA GG-3’ and Dp71R, 5’-TGC AGC TGA CAG GCT CAA GA-3’. Control immunoblot experiments were also performed on brain extracts to verify the Dp71-null phenotype. Because of the previously demonstrated influence of genetic background on cataract formation, and particularly due to beaded filament mutations and differential expressions, the cataract phenotype reported in this study was evaluated in the S129 original background as well as in mice backcrossed on either C57Bl/6 (pigmented) or Balb/c (albino) genetic backgrounds. All experiments were conducted in compliance with the European Communities Council Directives (86/609/EEC) for animal care and experimentation (HMG). Mice were handled in accordance with the ARVO statement for the use of animals in ophthalmic and vision research.

Clinical evaluation of mice eyes

Mice eyes were examined for lens opacity using a slit lamp and an ophthalmoscope. Mice were physically restrained during the analysis and their eyes were dilated with 1% tropicamide for at least 15 min before evaluation. Both wild-type (wt) (n = 20) and KO-Dp71 littermate mice (n = 31) were analyzed and mixed in a randomized order, without the evaluating investigators’ prior knowledge of the genotypes.

Electrophoresis, western blotting, and immunodetection

Retinas and lenses from 2-month-old mice were homogenized at 4 °C in 10 vol (wt/vol) of extraction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.01% sodium deoxycholate, 0.1% SDS) containing a mixture of protease inhibitors from Sigma (Saint-Quentin Fallavier, F), and it was centrifuged at 1,000 ×g for 5 min. Protein concentrations were determined by Bradford assays (Bradford, 1970). The proteins were separated using either NuPAGE Tris-Acetate 3%–8% or Tris-Glycine 4%–12% gradient gels (Invitrogen, Cergy Pontoise, F), and they were electrotransferred to either polyvinylidene difluoride membranes (Millipore, Saint-Quentin-en-Yvelines, F) or nitrocellulose (Schleicher & Schuell, Dassel, G), according to the manufacturer instructions. Blots were blocked with 5% dry milk (Bio-Rad, Hertfordshire, UK) in PBS for 1 h, and they were then incubated for 2 h with a primary antibody at room temperature. After washing, they were probed with an HRP-labeled goat anti-mouse secondary antibody (Interchim, France). Chemiluminescence was performed using the ECL+ plus western blotting detection system (Amersham Biosciences, UK), and it was documented on film (Kodak).

Lens histology, immunohistochemistry, and phalloidin staining

For a gross morphology analysis of the lenses, the dissected lenses were placed in a warm tissue culture Medium 199 containing Earle’s salts and l-glutamine (Cellgro, Mediatech, Inc., Manassas, VA). Transparency was then assessed using a dissecting microscope with photographing lenses placed on a 200-mesh electron microscopy (EM) grid using bright field conditions to optimize the focus on the grid, as previously described [15].

For histology, after enucleation, the eyes of 2-month-old mice were fixed for 1 h in 4% paraformaldehyde, transferred to PBS (pH 7.4), dehydrated in increasing concentrations of ethanol, and embedded in paraffin. Sections (8 µm) were stained with hematoxylin and eosin (H&E), and they were viewed by light microscopy or prepared for immunohistochemical analysis. Four to six individual images of the H&E-stained sections were combined for a full representation of the whole lens.

Immunohistochemical labeling was performed using the indirect immunofluorescence method on previously deparaffinized and rehydrated sections. After blocking in PBS (pH 7.4) containing 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.05% Triton X-100, sections were incubated with the primary antibody in PBS (pH 7.4) supplemented with 3% NGS, 1% BSA, and 0.1% Tween-20. Primary antibodies were detected using a secondary goat anti-mouse IgG antibody coupled to Alexa fluorophore (Molecular Probes, Eugene, OR). Cell nuclei were counterstained using DAPI, 1:500 in PBS (pH 7.4; Molecular Probes, Eugene, OR). Controls were prepared by omitting the primary antibody during incubation; in these controls, no specific staining could be detected.

Phalloidin staining was performed on isolated lenses from wt and KO-Dp71 mice. Freshly isolated lenses were fixed for an hour in 4% paraformaldehyde before being washed and permeabilized in a PBS (pH 7.4) solution containing 0.1% Triton X-100 for 15 min. Lenses were then incubated for 2 h in a solution of 1% BSA, 0.25% Triton X-100 containing Alexa488-labeled phalloidin, and Draq5 for nuclei visualization. Lenses were finally washed in 0.1% Triton X-100 and stored at 4 °C until image acquisition by confocal microscopy.

Antibodies

The mouse monoclonal Dys2 antibody used to detect dystrophins was from Tebu (Novocastra, Newcastle-on-Tyne, UK). The AQP0 and actin are mouse monoclonal antibodies from Abcys (Paris, F) and Millipore (Saint-Quentin-en-Yvelines, F), respectively.

Incidence of cataract formation in KO-Dp71 mice

Following the expression of the inserted reporter gene, the original characterization of the KO-Dp71 mice suggested that Dp71 was expressed in the crystalline lens [14]. To determine whether Dp71 plays a critical role in the crystalline lens, we assessed the lens transparency in wt and KO-Dp71 littermate mice strains at different ages post-development, including 2, 5, and 7 months of age. Although the initial characterization of KO-Dp71 mice shows that the gross anatomy of the eye appears normal (this study and 15), cataracts were observed in 37.5% of the 2-month-old KO-Dp71 mice. This proportion strongly increased in older mice to reach 100% of the 7-month-old mice tested (Figure 1A). In contrast and as expected, spontaneous cataracts were observed in only one of the 7-month-old wt littermates, thus indicating that the absence of Dp71 induces the development of juvenile congenital cataracts (Figure 1B). This result is consistent with the similar, but slower developing, congenital cataracts observed in the dystrophin mutant mdx3cv mice, which suffer a drastic reduction in—but not a disappearance of—all the DMD gene products (data not shown).

Histological analysis of lenses from KO-Dp71 mice

Gross morphological analyses of the isolated lenses of wt and KO-Dp71 mice showed that while lenses from the wt mice showed no morphological defects at the light level, an absence of Dp71 was clearly associated with refractive defects when placed on a 200-mesh EM grid (Figure 1). To better characterize the impact of the lack of Dp71 on the development and maintenance of a functional translucent lens, a histological analysis was performed on lenses from wt and KO-Dp71 mice at different ages. First, we observed that the size of the lens was consistently reduced in KO-Dp71 mice when compared with their wt littermates. This finding was maintained throughout life and represented here at 2 (65.2 mm³ in wt versus 36.8 mm³ in KO-Dp71) and 7 months (91.5 mm³ in wt versus 57.5 mm³ in KO-Dp71) to match the in vivo data (Figure 2). Careful observations of the wt lenses at these ages revealed normal lens development without cellular damage or disorganization in epithelial or fiber cells. While the overall structure and organization of the lenses were normal in the KO-D71 mice, consistent with the in vivo observations, subtle alterations were already detectable in lenses from 2-month-old KO-Dp71 mice, which was exacerbated at 7 months. At 2 months of age, a few instances of structural defects, such as the formation of vacuoles and a loss in the juxtaposition of fiber cells, were observed (Figure 2D, white arrows). Consistent with the slit-lamp observations, those defects seem to accumulate with age (Figure 2H, white arrows). Additionally, the distinct and typical bow-like organization of the cell nuclei observed in the wt mice began to be disrupted in 2-month-old KO-Dp71 mice. In KO-Dp71 animals, cell nuclei could be found much more centrally and deeply inside the fiber cell layers, well beyond the bow region of the equator. This was a phenotypic observation that was exacerbated with age, as demonstrated by images of lenses from 7-month-old KO-Dp71 mice (Figure 2H, arrowhead).

Comparative expression and localization of DMD gene products in lenses from wt and KO-Dp71 mice

The DMD gene product expression was assessed by western blot analysis on whole lens extracts from wt and KO-Dp71 mice, and they were compared with whole retina extracts. As previously reported, all the DMD gene products except Dp116 were expressed in mouse retinas and only the Dp71 expression was disrupted in the retinas of KO-Dp71 mice (Figure 3). An analysis of lenses from wt mice demonstrated that Dp140, Dp71, and Dp45 were expressed at high levels in this tissue, as well as that Dp71 seemed to be significantly more abundant than the others were. As expected, Dp71 was undetectable in lens lysate from KO-Dp71 mice, whereas the expression of Dp140 was unchanged (Figure 3). The loss of the Dp45 expression observed in the lenses—but not in the retinas—of KO-Dp71 mice suggests that the isoform expressed in the lens comes from the same promoter as Dp71. Alternatively, the protein expressed in the retina may be from a different promoter (Figure 3).

Discreet and specific localization of dystrophins and their associated complexes is critical for their role at the neuromuscular junction or in the central nervous system; thus, we assessed the specific cellular patterns of the expressions of dystrophins in lenses using immunohistochemistry. To this end, cross-sections of lenses from KO-Dp71 mice and their wt littermates were stained with a pan-specific antibody, recognizing all the DMD gene products. An analysis of the staining in tissues from wt animals revealed a wide expression throughout the lens, from the core to the epithelial cells (Figure 4C). When focusing on the anterior pole, the strongest staining was observed at the cell membranes of the secondary fibers; however, the basal side of the epithelial cells of the anterior pole was also positively stained for dystrophins (Figure 4). Of note, the latter was the only signal that remained when immunostaining was performed on KO-Dp71 mice (Figure 4G), suggesting that Dp71 was mainly localized in lens fiber cells, whereas the larger DMD gene products were present in lens epithelial cells. Higher magnification images clearly demonstrated the localization of dystrophin proteins at the membranes of the secondary fiber cells and its colocalization with AQP0 (Figures 4I–K).

AQP0 expression and localization in lenses of wt and KO-Dp71 mice

The progressive onset of cataracts combined with the histological findings suggested a progressive loss of structural integrity of the lens in the absence of Dp71. Because of its known central role in complexes necessary for the proper location of membrane transporters, channels, and receptors, we hypothesized that Dp71 could have a similar role in the lens. AQP0, also known as MIP-26, is a membrane-bound protein, abundantly and specifically expressed in lens fiber cells, and it regulates the transport of water. Neither the mRNA (data not shown) nor the protein levels of the AQP0 expression were significantly different between lenses from wt and KO-Dp71 mice (Figure 3). However, immunostaining against the AQP0 protein revealed a clear disorganization of the lens’ secondary fiber cells in the absence of Dp71 in both the anterior pole (Figure 4F) and the transition or germinative zone (Figure 5). Higher magnification images of the transition zones of KO-Dp71 mice lenses confirmed the relatively normal structural organization, but it also confirmed an increased number of nuclei deeper in the tissue (Figures 5G,I). A comparison of the AQP0 staining observed closer to the core of the lens also demonstrated more profound alterations in the structural organization of the lens fibers in the absence of Dp71 (Figures 5H,J). In addition to the vacuole-like structures also observed by histological analysis, staining for AQP0 revealed a disorganized pattern, an increased size of the fibers, and a more diffuse signal for AQP0, not as exclusively retained at the membrane as in wt animals.

Actin network in lenses of wt and KO-Dp71 mice

The DMD gene products, including Dp71, participate in multi-protein complexes that are important for the proper localization of transmembrane proteins, such as ion and water channels, through their interactions with the extracellular matrix on one side and the actin cytoskeleton on the other. Since the integrity of the actin cytoskeleton is a key factor in keeping the lens transparent, we analyzed how it was affected by the absence of Dp71.

Consistent with the limited impact of the absence of Dp71 observed through other methods, an analysis of the actin filaments in the superficial epithelial cells did not reveal any profound alterations in KO-Dp71 mice, even at 9 months of age (Figures 6A–D). Interestingly, however, consistent with the alterations observed through histology and immunostaining, the actin filaments were noticeably disorganized starting at 3 months (Figure 6F), an effect that was enhanced at 9 months of age (Figure 6H).