p62/Sequestosome 1 levels increase and phosphorylation is altered in Cx50D47A lenses, but deletion of p62/sequestosome 1 does not improve transparency

Antibodies

Rabbit polyclonal anti-p62/SQSTM1 antibody (P0067; lot # 126M4796V) was obtained from Sigma (St. Louis, MO). Rabbit polyclonal anti-phospho-SQSTM1/p62 (Thr269/Ser272) (13121S; lot # 2), anti-phospho-SQSTM1/p62 (Ser349) (95697S; lot # 1), and anti-histone H3 (4499S; lot # 9) were obtained from Cell Signaling Technology (Danvers, MA). Polyclonal anti-Ubiquilin1 (PA5-22174; lot # UL2903952C) was obtained from Thermo Fisher Scientific (Waltham, MA). Mouse monoclonal anti-Ubiquilin2 (MABN763; lot # 3035872) was obtained from MilliporeSigma (Burlington, MA). Mouse monoclonal anti-Ubiquilin4 (sc-136145; lot # E1716) and rabbit polyclonal anti–β-crystallin (sc-48335; lot # A1706) and anti-translocase of outer mitochondrial membrane 20 (TOM20) (sc-11415; lot # D1613) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Affinity-purified rabbit polyclonal anti-Cx46 intracellular loop and anti-Cx50 C-terminus antibodies have been previously described [6,21]. Other primary antibodies (to cellular compartments and crystallins) and secondary antibodies were the same as those used in other recent studies from our laboratory [22,23]. Rabbit polyclonal anti–αA (SPA-221; lot # 10010805) and anti–αB-crystallin (SPA-223; lot # 08011134) antibodies were obtained from Enzo Life Sciences (Farmingdale, NY). Rabbit polyclonal anti–γ-crystallin antibodies were a kind gift from Dr Samuel Zigler (Wilmer Eye Institute at Johns Hopkins University, Baltimore, MD). Horseradish peroxidase–conjugated goat AffiniPure anti-rabbit immunoglobulin G (IgG; H+L; 111–035–144; lot # 123520 and 135724) and horseradish peroxidase–conjugated goat AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment-specific (115–036–072; lot # 111686 and 135721) antibodies were obtained from Jackson ImmunoResearch (West Grove, PA).

Animals

Cx50D47A (also known as No2 or ENU-326) mice [4] were maintained in the C3H line as previously described [6]. p62 null mice [24] were obtained from Dr. Toru Yanagawa (University of Tsukuba, Tsukuba, Japan). These p62 knockout mice were bred into the C3H line for six generations before the experiments were performed. Microscopic examination of the lenses of these animals in our laboratory showed no apparent differences in size or transparency compared to wild-type mice of the same strain. Heterozygous p62 null (p62^+/−) mice were mated with homozygous Cx50D47A (Cx50^D47A/D47A) mice to generate heterozygous p62 knockout mice that were homozygous for the Cx50 mutation (p62^+/−-Cx50^D47A/D47A). Mice of this genotype from different litters were mated to obtain homozygous Cx50D47A mice that were wild-type for p62 or heterozygous or homozygous for p62 deletion. All animal procedures followed the University of Chicago Animal Care and Use Committee guidelines and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the National Institutes of Health regulations.

Quantification of lens sizes and opacities

Dark-field photomicrographs of lenses from 1-month-old mouse littermates containing all genotypes (referred to as a set) were obtained using a Zeiss Stemi-2000C dissecting scope (Carl Zeiss, München, Germany). The same settings (i.e., magnification, illumination, and exposure time) were used to photograph all lenses within each set. Cataracts (opacities) were quantified in photomicrographs by integrating the gray values over a circular region of interest (of identical size for all images) using NIH ImageJ software (https://imagej.nih.gov/ij/) as previously performed [8,23]. These integrated opacity data are reported in arbitrary units. Equatorial diameters were determined from the lens photomicrographs using NIH ImageJ software. The opacities and the diameters were measured in both lenses of each mouse (unless one of the lenses was damaged during dissection), and the reported values are the averages of the measurements of the two lenses for each mouse.

Staining of lens nuclei

Lenses from 1-month-old mice were fixed in 4% paraformaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4). After several rinses, the lenses were transferred to 30% sucrose in PBS and left at 4 °C until they sank. Cryostat sections (18 µm) were obtained and incubated with 4′,6-diamino-2-phenylindole dihydrochloride (DAPI; Invitrogen, Carlsbad, CA) to stain the nuclei. Specimens were observed using an epifluorescence Axioplan 2 microscope (Carl Zeiss). Images (10X magnification) were acquired with a Zeiss AxioCam digital camera using Zeiss AxioVision software. Photomontages were constructed, and composite figures were assembled using Adobe Photoshop CS3 Extended (Adobe Systems, Inc., San Jose, CA).

Immunoblotting

Proteins were detected and quantified with immunoblotting of whole lens homogenates prepared from the two lenses of 1-month-old mice as described by Minogue et al. [23]. Samples were prepared by homogenizing the 2 lenses from each mouse in PBS containing 4 mM EDTA, 2 mM phenylmethyl sulfonylfluoride, 20 mM NaF, 10 mM Na₃VO₄ and cOmplete EDTA-free protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) using a glass-glass homogenizer followed by sonication. Protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA). Aliquots from lens homogenates containing equal amounts of total protein were loaded in each lane and resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels. Gels were then blotted to Immobilon P membranes (Millipore, Bedford, MA) using a wet transfer apparatus. Then, the membranes were stained with Ponceau S (to verify equal electrotransfer of the proteins) before being subjected to immunoblotting as previously described [7,23]. The bands obtained in at least three independent experiments (i.e., biological replicates) were quantified by densitometry using a rectangular box that encompassed the immunoreactive band(s) using Adobe Photoshop CS3.

Statistical analysis

Raw data obtained from heterozygotes and homozygotes were compared to the raw data obtained from wild-type (or control) littermates to assess statistical significance using the Student t test. Statistical analysis was performed using Microsoft Excel. Graphs were prepared using SigmaPlot 10.0 (Systat Software, Inc.). A p value of less than 0.05 was considered statistically significant. The sets of littermates containing all genotypes (statistical n) totaled at least three for each type of data presented.

Levels of p62 and phosphorylated p62 are altered in homozygous Cx50D47A lenses

We used immunoblotting to examine the levels of total p62 in lenses of wild-type mice and mice that were heterozygous or homozygous for the Cx50D47A mutation (Figure 1A–C). Quantitation of blots showed that total p62 levels in lens homogenates did not differ significantly between wild-type and Cx50D47A heterozygotes, but the total p62 levels were more than twice as high in homozygotes (Cx50^D47A/D47A) as in wild-type mice (Figure 1D).

We also used immunoblotting to assess the levels of p62 phosphorylated at T269/S272 (P-p62(T269/S272)) and p62 phosphorylated at S349 (P-p62(S349)) in these lenses (Figure 1B,C). Although the ratio of P-p62(T269/S272) to total p62 did not differ between lenses from wild-type mice and mice that were heterozygous for the Cx50D47A mutation, the ratio was substantially and significantly reduced in the lenses of homozygotes (Figure 1E). In contrast, the ratio of P-p62(S349) to total p62 was significantly increased in lenses from homozygous Cx50D47A mice compared to that for the wild-type animals (Figure 1F); this ratio was not significantly different between wild-type and heterozygous Cx50D47A lenses (Figure 1F). Thus, there is a specific decrease in phosphorylation of p62 at T269 or S272 and a specific increase in its phosphorylation at S349 in the homozygous Cx50 mutant lenses.

Deletion of p62 expression did not affect size or transparency of homozygous Cx50D47A lenses

To test the importance of p62 in the reduced growth and cataract formation in Cx50D47A lenses, we generated litters of mice that were homozygous for the Cx50D47A mutant allele and contained all three p62 genotypes (wild-type and heterozygous and homozygous p62 null) by breeding with p62 null mice (which do not show lens abnormalities). We used immunoblotting to confirm the knockout of p62 in these animals. The immunoblots showed that p62 levels were significantly reduced (by more than 50%) in lenses of heterozygotes (p62^+/−-Cx50^D47A/D47A) compared with lenses from mice that were wild-type for p62 (p62^+/+-Cx50^D47A/D47A). p62 levels were undetectable in the lenses of homozygotes (p62^−/−-Cx50^D47A/D47A; Figure 2).

We examined the size, appearance, and transparency of the lenses of these mice by obtaining photomicrographs using dark-field illumination (Figure 3). All of these lenses appeared similar and contained cataracts regardless of their p62 genotype (Figure 3A). The lenses did not differ in size, based on measurements of equatorial diameters (Figure 3B). There were no significant differences in the integrated densities of opacities between the cataracts in the lenses from p62^+/+-Cx50^D47A/D47A, p62^+/−-Cx50^D47A/D47A, and p62^−/−-Cx50^D47A/D47A mice (Figure 3C). Thus, deletion of p62 did not lead to a significant improvement or worsening of the growth or opacification of homozygous Cx50D47A lenses.

Deletion of p62 did not affect levels of lens fiber connexins in Cx50D47A mice

Among the intracellular functions of p62, it modulates different proteolytic pathways. Because Cx50D47A mice have low levels of both lens fiber connexins, we suspected that p62 might contribute to their degradation. To test this hypothesis, we evaluated the effects of p62 deletion on levels of Cx46 and Cx50 by performing immunoblots for Cx46 (Figure 4A) and Cx50 (Figure 4B) on whole lens homogenates from p62^+/+-Cx50^D47A/D47A, p62^+/−-Cx50^D47A/D47A, and p62^−/−-Cx50^D47A/D47A mice. Quantification of the levels of immunoreactive Cx46 (Figure 4A, bottom panel) and Cx50 (Figure 4B, bottom panel) did not show any significant differences between the Cx50D47A mice regardless of the p62 genotype.

Deletion of p62 did not affect the levels of nuclear or mitochondrial proteins in the lenses of Cx50D47A mice

Because the lenses of Cx50D47A mice contain an increased abundance of nuclei and cellular organelles that are normally degraded during lens differentiation, we hypothesized that the elevation of p62 and the specific increase in its phosphorylation at S349 might reflect stimulation of autophagy in response to their impaired degradation. We determined the levels of histone H3 (one of the major proteins found in the chromatin of eukaryotic cell nuclei) and the mitochondrial import receptor subunit TOM20 homolog in whole lens homogenates of homozygous Cx50D47A mice that were wild-type for p62 or heterozygous or homozygous for the deletion of p62 (Figure 5A-D). Quantitation of the immunoblots showed that levels of histone H3 and TOM20 did not differ significantly between the p62 genotypes (Figure 5C,D). We also stained sections of p62^+/+-Cx50^D47A/D47A, p62^+/−-Cx50^D47A/D47A, and p62^−/−-Cx50^D47A/D47A lenses with DAPI to detect nuclei and nuclear fragments; the DAPI staining did not appear different between the p62 genotypes (Figure 5E).

Deletion of p62 did not alter the levels of crystallins in Cx50D47A lenses

Because p62 could be involved in the altered levels of crystallins that occur in many types of cataracts, including mice with connexin deletions or mutations [6,22,25], we examined levels of several crystallins by immunoblotting of lens homogenates (Figure 6). We found no significant differences in the levels of αA-, αB-, β-or γ-crystallins among the p62^+/+-Cx50^D47A/D47A, p62^+/−-Cx50^D47A/D47A, and p62^−/−-Cx50^D47A/D47A lenses (Figure 6).

The lack of effect of p62 deletion could result from altered ubiquilin expression

Other proteins, like the ubiquilins, that act as adaptors and link proteolytic pathways [26] could potentially compensate for the lack of p62. Therefore, we performed immunoblots of whole lens homogenates to detect the levels of ubiquilins 1, 2, and 4 (UBQLN1, UBQLN2, and UBQLN4) [27].

We initially studied animals that were wild-type for p62 (p62^+/+) and had different Cx50D47A genotypes. The lenses of the heterozygous (Cx50^+/D47A) and homozygous Cx50D47A mice (Cx50^D47A/D47A) showed a decrease in UBQLN1 levels compared to wild-type mice; however, this difference reached statistical significance only for homozygotes that showed a 39% reduction in UBQLN1 levels (Figure 7A). The levels of UBQLN2 or UBQLN4 were not significantly different between the lenses of the Cx50^+/+, Cx50^+/D47A, and Cx50^D47A/D47A mice (Figure 7 B,C).

Then, we studied mice that were homozygous for the Cx50D47A mutation and had different p62 genotypes. Heterozygous or homozygous deletion of p62 did not affect the levels of UBQLN1 in the homozygous Cx50D47A lenses (Figure 7D). However, the levels of UBQLN2 were increased in the Cx50D47A lenses with homozygous (but not with heterozygous) deletion of p62 (Figure 7E); it represented an 84% increase above the levels in the lenses from the Cx50D47A mice that were wild-type for p62 (p62^+/+-Cx50^D47A/D47A; Figure 7E). The levels of UBQLN4 were not significantly different among the homozygous Cx50D47A lenses of different p62 genotypes (Figure 7F).