CRYβA3/A1-Crystallin Knockout Develops Nuclear Cataract and Causes Impaired Lysosomal Cargo Clearance and Calpain Activation

<div><p>βA3/A1-crystallin is an abundant structural protein of the lens that is very critical for lens function. Many different genetic mutations have been shown to associate with different types of cataracts in humans and in animal models. βA3/A1-crystallin has four Greek key-motifs that organize into two crystallin domains. It shown to bind calcium with moderate affinity and has putative calcium-binding site. Other than in the lens, βA3/A1 is also expressed in retinal astrocytes, retinal pigment epithelial (RPE) cells, and retinal ganglion cells. The function of βA3/A1-crystallin in the retinal cell types is well studied; however, a clear understanding of the function of this protein in the lens has not yet been established. In the current study, we generated the βA3/A1-crystallin knockout (KO) mouse and explored the function of βA3/A1-crystallin in lens development. Our results showed that βA3-KO mice develop congenital nuclear cataract and exhibit persistent fetal vasculature condition. At the cellular level KO lenses show defective lysosomal clearance and accumulation of nuclei, mitochondria, and autophagic cargo in the outer cortical region of the lens. In addition, the calcium level and the expression and activity of calpain-3 were increased in KO lenses. Taken together, these results suggest the lack of βA3-crystallin function in lenses, alters calcium homeostasis which in turn causes lysosomal defects and calpain activation. These defects are responsible for the development of nuclear cataract in KO lenses.</p></div

Slit-lamp examination of lenses of WT, HET and KO.

<p>4-weeks-old mice were examined with a Micron IV-slit lamp after dilation of the pupils. <b>i)</b>. Slit-lamp images of the WT, HET and KO mice eyes from the front view. Clear nuclear cataracts were observed in the center (nuclear region) of the KO lenses (n = 3 animals). <b>ii)</b>. Images of the lenses with the slit-beam incident on the side of the eye. Images showing the presence of a fibrous mass (shown by an arrow), fetal hyaloid artery retention (condition called Persistent Fetal Vasculature) posterior to the lens. <b>iii)</b>. The Y-shaped suture line is distorted in KO lenses compared to a distinct Y-shaped suture line in WT and HET lenses. <b>iv)</b>. Illumination from the back of the lens showing the attachment of the hyaloid artery in a KO lens. (n = 3 animals).</p

Mitochondria accumulate in CRYβA3/A1KO mice.

<p>Transmission Electronmicrographs 1-months old WT, HET and KO lenses. <b>A</b>. EM images of lens epithelium. The epithelium of KO lenses shows a number of double-membraned vesicles containing undegraded organelles, mostly mitochondria (shown by a black arrow, and also in the enlarged inset image). No such accumulation of mitochondria in the epithelium of WT or HET lenses was seen. <b>B</b>. EM images of the equatorial region of the lens. The fiber cells in KO lens show presence of mitochondria and other organelles as shown by red arrows and in enlarged inset image. <b>C</b>. EM images of the nuclear region (center) of the lens. The nuclear regions of WT and HET lenses were devoid of any cytoplasmic debris (indicated by arrows and in enlarged inset image). Cytoplasmic debris were present in the center of the KO lens (indicated by an arrow and enlarged inset image). n = 2 independent experiments. Scale bars: 2 μm and inset images 500 nm.</p

Nuclear degradation process is impaired in CRYβA3/A1KO mice.

<p><b>A</b>. H&E staining of 1-month old WT, HET and KO mice lens sections. An arrow in KO lens image show an abnormally high number of nuclei compared to WT and HET lenses. <b>B</b>. DAPI-staining of 1-month old lens sections of WT, HET and KO mice. DAPI-staining shows an accumulation of nuclei and nuclear debris in the OFZ (organelle free zone) and in nuclear region of KO lenses (indicated by an arrow). The bar graph on the right shows the quantification of nuclei. Data represent mean ± S.D in n = 4 independent experiments. **, P<0.01 by student’s t test. Scale bars: A: 100 μm, B: 50 μm.</p

Calpain-3 is activated in KO lenses.

<p><b>A</b>. Immunostaining of 1-month old lens sections with anti-calpain Lp-85 antibody. KO lens show an increased expression of calpain compared to WT. Inset images a and b are enlarged areas of the equatorial region of WT and KO lens sections respectively, demonstrating positive calpain staining in the nuclei in KO compared to WT lenses. <b>B</b>. Cultured LECs immunostained with anti-calpain Lp-85 antibody. KO cells clearly show calpain staining on the nucleus, compared to WT cells that show calpain staining around the nucleus. Inset images are enlarged areas to visualize the nuclear staining. Data is representative of 6–8 cells for each type. Scale bars: A&B: 50 um and a, b, c and inset images: 20 um. <b>C</b>. Determination of calpain activity using Suc-LLVY-AMC calpain substrate. Calpain activity was plotted as relative light units/mg of protein. **, P>0.01 students t test.</p

Integrity of cytoskeletal network is affected in KO lenses.

<p>Expression of alpha spectrin is altered in KO. <b>A</b>. SDS-PAGE showing the protein profile of 1-month old WT and KO lens homogenates. The expression of 120 kDa and 240 kDa protein band was increased in 1-month old KO lenses (shown by an arrow) n = 5 independent experiment. <b>B</b>. Identification of 120 kDa protein as a fragment of alpha spectrin by mass spectrometry. <b>C</b>. Western blot analysis of 1-month old lens homogenates probed with anti-alpha spectrin antibody showing an increased degradation of alpha spectrin in KO lenses. n = 2 independent experiment. <b>D</b>. Expression pattern of alpha-II spectrin in LECs. Immunostaining of LECs with alpha-II spectrin antibody showing the cytoskeletal meshwork of α-spectrin in WT LECs. In contrast, in KO LECs the α-spectrins meshwork are aggregated. Data is representative of 6–8 cells for each type. Scale bars: 20 μm. <b>E</b>. LECs were treated with F-actin phalloidin to visualize the actin polymerization. KO LECs showed a reduction in F-actin staining compared to WT LECs. n = 2 independent experiment. Scale bars: 20 μm. <b>F</b>. Western blots of 1-month-old lens homogenates probed with a β-actin antibody. Compared to WT the expression of β-actin was not altered in KO lenses. <b>G</b>. Immunostaining of LECs with the <b>α</b>-tubulin antibody. WT and cells show well-spread tubulin strands compared to KO LECs. In KO cells tubulin strands are aggregated, and the aggregation is prominent near the edges (shown by arrow and inset panel; enlarged area). Data is representative of 6–8 cells for each type Scale bars: 20 μm. <b>H</b>. Western blots of 1-month old lens homogenates probed with <b>α</b> -tubulin antibody showing an unchanged level of <b>α</b>-tubulin protein in KO lenses.</p

Ca²⁺ level is altered in KO cells.

<p>Cultured LECs from WT, and KO lenses were incubated with a Ca²⁺- dye, Calcium Orange (4 μM; red) for 45 minutes, during which dye enters intracellular organelles. Fluorescence intensity was higher in KO cells than in the WT cells. Intense fluorescent signal was localized around the nucleus in KO (b) compared to diffused cytoplasmic staining in WT cells (a). (Data is representative of 8–10 cells for each type and n = 2 independent experiment). Scale bars: 20 μm. Bar graph on the right is the quantification of calcium signal. *, P>0.05 by students t test.</p

Generation of CRYβA3/A1 knockout mice.

<p><b>A</b>. Organization of the CRYβA3 gene on chromosome 11, consisting of 6 exons flanked by introns. Exon 3–6 are the critical exon that encode four Greek key-motifs I-IV. <b>B</b>. Schematic of the first knockout strategy used for generating βA3/A-crystallin KO mice. Shown is the Cryba1^{tm1a(EUCOMM)Hmgu} “knockout first” allele with a (FRT)-flanked selection cassette inserted into the third intron of the βA3/A1 gene, which traps the transcript through the Engrailed-2-splice acceptor (En2 sA) and truncates it through the SV40 polyadenylation signal. Primer specific to the neomycin resistance gene was used to confirm the disruption of the βA3/A1 gene. <b>C</b>. Genotyping result for WT, HET and HOM KO mice. <b>D</b>. Confirmation of the deletion of βA3/A1 mRNA in KO lenses by qRT-PCR. <b>E</b>. Western blot analysis using a polyclonal βA3/A1-crystallin antibody. Lack of band at 50 kDa dimer position (βA3-crystallin normally exist as a dimer) in KO lenses and reduced intensity in HET lenses.</p

Autophagic Markers Accumulate in CRYβA3/A1 KO mouse.

<p>Detection of LC3 levels in KO mice lenses. <b>A</b>. Western blot of 1-months old lens homogenates probed with LC3-antibody (red) and GAPDH (green). Compared to WT and HET lenses, the ratio of LC3-II to LC3-I was greater in KO lenses. The bar graph below is the quantification of relative ratio of LC3-II to LC3-I. Data represent mean ± S.D in n = 3 animals. *, P<0.05 by student’s t test. <b>B</b>. Immunohistochemical staining of 1-month-old lens sections with the LC3 antibody. (LC3: red, DAPI nuclear stain: blue) Intensity of LC3 staining was increased, 2–3 folds in KO lenses compared to WT and HET lenses. The OFZ of KO lenses (panel c) showed positive LC3 staining compared to the corresponding region in WT (panel a) and HET (panel b) lenses. Bar graph on the right panel represent quantification of LC3 staining (mean ± S.D) in n = 4 lenses. *, P<0.05 by student’s t test. <b>C</b>. Cultured Lens Epithelial Cells (LECs) from WT, HET and homozygous KO mice were examined by immunofluorescence using an anti-LC3 antibody (green). <b>D</b>. Analysis of p62/SQSTM expression in KO mice lenses. Western blot of 1-months lens homogenates probed with the p62-antibody (white) and GAPDH antibody (green). The bar graph on the right shows the quantification of p62 intensity. Data represent mean ± S.D in n = 3 animals. *, P<0.05 by student’s t test. <b>E</b>. Immunohistochemical analysis of p62 distribution in mouse lens sections. LEC and lens fiber cells of the KO show increased p62 expression (red) and larger p62 aggregates compared to the LEC and lens fiber cells of WT and HET lens. The bar graph on the right shows the quantification of p62 intensity. Data represent mean ± S.D in n = 4 lenses. *, P<0.05 by student’s t test. <b>F</b>. Immunofluorescence analysis of p62 in LEC. KO cells show increased expression of p62 and larger sized p62 aggregates compared to HET and WT cells. n = 2, independent experiments. <b>G</b>. Western blot of lens homogenates probed with the ubiquitin antibody (green) and GAPDH antibody (white). The bar graph on the right shows the quantification of ubiquitin signal. Data represent mean ± S.D in n = 3 animals. *, P<0.05 by student’s t test. <b>H</b>. Immunohistochemical analysis of ubiquitinated proteins in mouse lens sections. Outer cortical region of KO showing drastic increase in ubiquitinated proteins (red) compared to control lenses. Bar graph on the right side is a quantification of ubiquitin signal represented as mean ± S.D in n = 3 lenses. *, P<0.05 by student’s t test. Scale bars: B: 100 um, a,b and c, E, H: 50 μm, and C, F:20 μm.</p

Visulaization of colocalization of lysosomes with autophagosomes.

<p>Lens epithelial cells from WT and KO lenses were cultured and immunostained with anti-Lamp-II antibody (red) to visualize lysosomes followed by staining with LC3B/MAP1LC3B antibodyconjugated with AlexaFluor 488 (green) and DAPI nuclear stain (blue) to label autophagosomes and nuclei respectively. Panels (A-D) show immunostaining of WT lens epithelial cells and (E-H) of epithelial cells from KO mice lenses. (Data is representative of 8–10 cells for each type). Scale bars: 20 μm.</p