The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses*

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo. Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

Proteins that do not traffic properly or are misfolded can cause endoplasmic reticulum (ER) 2 stress. To compensate for the disturbance of proteostasis, cells transduce ER stress through at least three different signaling pathways: (a) the inositol-requiring enzyme 1 (IRE1␣)-dependent, (b) the activating transcription factor 6 (ATF6)-dependent, and (c) the PRKR-like ER kinase (PERK)-dependent pathways. Prolonged and uncompensated ER stress triggers the unfolded protein response (UPR), which may ultimately target the cells for apoptosis.
Many disease-linked connexin mutants show impaired cellular trafficking. One such mutant has a missense mutation in the lens gap junction protein, connexin50 (Cx50), affecting amino acid residue 47 (Cx50D47N in humans and Cx50D47A in mice) and is linked to congenital cataracts (1,2). When expressed in transfected cells, Cx50D47N and Cx50D47A localize mostly within the ER (1). Lenses from mice expressing Cx50D47A have nuclear cataracts, reduced sizes, and impaired denucleation (3). Some immunoreactive Cx50 localizes intracellularly in lens epithelial cells from these mice (3). Homozygous Cx50D47A lenses have increased levels of ␣B-crystallins (3), which are responsive to several types of stress in different cell types, including lens epithelial cells (4 -7). These observations suggest that expression of a Cx50D47 mutant may cause ER stress in the lens and contribute to pathology in this organ.
The current understanding of cellular responses to ER stress is based on many studies in cultured cells and fewer studies in vivo. Although massive induction of ER stress by pharmacological agents may activate all three transducing pathways, the UPR is a regulated pathway that may differentially use the transducing pathways, depending on the stimulus causing the ER stress. To elucidate the effects of in vivo expression of a connexin with impaired trafficking, we studied the Cx50D47A mouse and examined the activation of each of the ER stresstransducing signaling pathways in the lens. We found evidence for activation of the PERK-dependent pathway, but not of the IRE1␣or ATF6-dependent pathways, in Cx50D47A-expressing lenses.

Results
The Ire1␣-dependent Pathway Is Not Activated in 1-Month-old Cx50D47A Mice-Activation of the IRE1␣ pathway leads to splicing of its downstream target, Xbp-1 (X-boxbinding protein 1) RNA. To test whether this ER stress-transducing pathway was activated in Cx50D47A-expressing lenses, we determined the relative levels of spliced Xbp-1 transcripts on the lens by RT-qPCR. Although variations in the levels of spliced Xbp-1 transcripts were detected among individuals (even within each genotype), they correlated with proportional changes in mRNA levels for total Xbp-1. Indeed, the spliced/ total ratios of Xbp-1 transcripts in samples from heterozygous and homozygous animals were similar to those in wild type mice (Fig. 1), suggesting that this pathway is not activated by expression of Cx50D47A. As a positive control, a wild type lens was treated with thapsigargin, a non-competitive inhibitor of the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), which induces the UPR pharmacologically. The spliced/total ratio of Xbp-1 transcripts in this sample was 3.8 ( Fig. 1).
The ATF6␣-dependent Pathway Is Not Activated in Cx50D47A Mice-ER stress-induced activation of the ATF6 pathway leads to translocation of ATF6␣ from the ER to the Golgi compartment, where it is cleaved by SP1 and SP2 (8), resulting in a change in apparent molecular mass from 90 to 50 kDa (9). To test whether expression of Cx50D47A led to activation of the ATF6-dependent transducing pathway, whole lens homogenates were subjected to immunoblotting with anti-ATF6␣ antibodies. Although the antibody recognizes both the full-length and cleaved forms of ATF6␣, only the cleaved form was detected in lens samples (Fig. 2). Surprisingly, levels of cleaved ATF6␣ were significantly decreased in both heterozygous and homozygous Cx50D47A mice, respectively, representing on average 37.6 and 6.8% of the values detected in wild type littermates (Fig. 2), suggesting deactivation of this pathway in the mutant mice.
The PERK-dependent Pathway Is Activated by Expression of Cx50D47A-Activation of the PERK-dependent signaling pathway involves PERK autophosphorylation. To test whether Cx50D47A led to activation of this pathway, we performed immunofluorescence on lens sections using an antibody that specifically detects the active (phosphorylated) form of PERK (P-PERK). Most of the P-PERK immunoreactivity appeared as small puncta located in epithelial and fiber cells near the bow region (Fig. 3, A-F). A significant increase in immunoreactive P-PERK was detected in sections from heterozygous and homozygous Cx50D47A mice as compared with wild type animals (Fig. 3G), but the increase did not reach statistical significance between heterozygous and homozygous lenses. Because activation of PERK leads to phosphorylation of EIF2␣, we performed immunoblots to determine whether it occurred in Cx50D47A-expressing lenses. Heterozygous lenses showed a significant 1.4-fold increase in the relative ratio of phosphorylated to total EIF2␣ (P-EIF2␣/EIF2␣) above the ratio in wild type lenses, and homozygous lenses showed a significant 2.3fold increase over the wild type ratio (Fig. 3H). Because phosphorylation of EIF2␣ may lead to increased translation of ATF4 (activating transcription factor 4 (CREB2)), we determined its levels in the lens. Levels of ATF4 were significantly increased in lenses from homozygous Cx50D47A mice, representing on average 391% of the levels detected in wild type mice, whereas levels of ATF4 in heterozygous lenses were similar to those of wild type animals (Fig. 3I).
To test whether expression of Cx50D47A altered transcription or levels of ER proteins, we performed RT-qPCR and immunoblotting. Protein levels of the calcium-binding chaperone, calreticulin, which is mainly localized in the ER, were significantly increased in Cx50D47A-expressing lenses; they represented on average 184% in heterozygous and 262% in homozygous lenses of the calreticulin levels in wild type littermates (Fig. 4A). Levels of GRP78 (also known as BIP) were significantly increased in heterozygous and homozygous lenses (146 and 164%, respectively) relative to the levels in wild type lenses (Fig. 4B). The mRNA levels for two other ER-resident proteins that are often increased in the UPR, p58 IPK (a co-chaperone of heat shock 70-kDa protein 8) and Erp72 (a proteindisulfide isomerase), were similar among all genotypes (Fig. 4, C and D).
In the canonical ER stress response, increased transcription of CHOP is downstream from ATF4. To test whether the FIGURE 1. Expression of Cx50D47A does not activate the IRE1␣-dependent pathway. The graph shows the ratio of spliced (sXbp-1) over total (tXbp-1) Xbp-1-fold changes for 1-month-old Cx50D47A heterozygous (ϩ/D47A) and homozygous (D47A/D47A) lenses relative to wild type (ϩ/ϩ) littermates as determined by RT-qPCR obtained in three independent experiments (black circles). The bars represent the arithmetic means of the ratios for each genotype. The short dashed line marks the reference value of wild type mice (sXbp-1/tXbp-1 ϭ 1.0). A thapsigargin-treated wild type lens (ϩ/ϩ Tg) was used as a positive control; the change in its sXbp-1/tXbp-1 ratio is shown on the right. increased levels of ATF4 resulted in alterations in CHOP, we determined its transcript and protein levels. We detected significant increases of Chop mRNA in heterozygous and homozygous lenses (1.9-and 12.4-fold on average, respectively) compared with the levels detected in wild type lenses (Fig. 5A). A CHOP-immunoreactive band was clearly visible in immunoblots of samples from Cx50D47A homozygous mice, but this band was only barely detectable in heterozygous and wild type samples, implying that overall levels of the CHOP protein are extremely low in the normal lens (Fig. 5B). Levels of CHOP in homozygotes represented on average 384% of wild type levels (p Ͻ 0.05), whereas CHOP levels in heterozygotes were on average 73% of wild type levels (p ϭ 0.3). To determine the cellular compartment of CHOP localization, we performed immuno-  4). H, immunoblots of immunoreactive phosphorylated (top) and total (bottom) EIF2␣ in total lens homogenates from 1-month-old wild type (ϩ/ϩ) and Cx50D47A heterozygous (ϩ/D47A) and homozygous (D47A/D47A) mice. Fifteen g of total protein were loaded in each lane to detect phosphorylated EIF2␣; 60 g of total protein were loaded per lane to detect total EIF2␣. The migration positions of the molecular mass markers are indicated on the left. The densitometric values of the bands obtained in three independent experiments were used to calculate the phosphorylated/total EIF2␣ ratios (P-EIF2␣/EIF2␣). The graph shows the P-EIF2␣/EIF2␣ relative to the value obtained in wild type littermates (black circles). I, immunoblot of immunoreactive ATF4 in aliquots of total lens homogenates from 1-month-old wild type (ϩ/ϩ), and Cx50D47A heterozygous (ϩ/D47A) and homozygous (D47A/D47A) mice containing 100 g of total protein. The migration positions of the molecular mass markers are indicated on the left. The graph shows the densitometric values of the bands obtained in three independent experiments expressed as percentages of the values obtained in wild type animals (black circles). The bars in G-I indicate the average value for each genotype. Asterisks denote significant differences from wild type values (p Ͻ 0.05). AUGUST  fluorescence. Immunoreactive CHOP staining was localized to nuclei of cells in the epithelium and bow region of all genotypes and in wild type lenses treated with thapsigargin in vitro to induce UPR (Fig. 5, C-T). Lenses from wild type (Fig. 5, C, F, and I) or Cx50D47A homozygous (Fig. 5, E, H, and K) mice showed much weaker CHOP immunoreactivity than wild type lenses treated with thapsigargin ( Fig. 5, D, G, and J), suggesting that the endogenous ER stress induced by expression of Cx50D47A is milder than an abrupt pharmacologically induced ER stress.

Cx50D47A Causes ER Stress in the Lens
Because increased CHOP can lead to apoptosis, we performed TUNEL staining on lens sections. In 1-month-old wild type lenses, fluorescent puncta were detected within a few cells at the border of the organelle-free zone (where they often colocalized with DAPI-stained material probably representing cells undergoing denucleation, which is part of the normal process of fiber cell differentiation), as expected from previous studies of normal lenses (10). Less frequently, fluorescent puncta (that did not co-localize with DAPI-stained material) were detected in cells within the organelle-free zone (Fig. 6, A-C, J, and K). In contrast, within homozygous Cx50D47A lens sections, many TUNEL-stained puncta were detected that colocalized with some nuclei and many nuclear remnants (i.e. DAPI-stained material of smaller size and different morphology than the intact nuclei in epithelia and early differentiating fiber cells), most of which localized toward the center of the lens (Fig. 6, G-I). Sections from Cx50D47A heterozygous lenses also showed an increase in TUNEL-stained puncta compared with wild type lenses but less than that seen in homozygotes (Fig. 6, D-F). Examination of these samples at higher magnification revealed low intensity TUNEL-fluorescent puncta in the Asterisks denote a significant difference as compared with wild type animals (p Ͻ 0.05). AUGUST 19, 2016 • VOLUME 291 • NUMBER 34 epithelia and differentiating fiber cells from Cx50D47A heterozygous and homozygous lenses that were seldom detected in wild type lenses (Fig. 6, J-O). Quantitative analyses of integrated density revealed a significant increase in TUNEL staining in heterozygous and homozygous samples compared with wild type (Fig. 6Q). When considered as ratios, the integrated densities of TUNEL staining were 19.6 times (heterozygotes) and 371 times (homozygotes) that of the wild type. These results presented a conundrum. Activation of the PERK-dependent UPR pathway (with increase in ATF4 and CHOP levels) in Cx50D47A homozygotes should have resulted in severe apoptosis and cell death. However, not all lens cells showed signs of DNA fragmentation after TUNEL staining. This prompted us to look for factors that might increase cell survival. Transcripts for Trib3 (Tribbles pseudokinase 3) and Irs2 (insulin receptor substrate 2) have been identified as prosurvival genes in early stage erythroblasts (which lose their nuclei during differentiation like lens cells) (11). To test whether expression of Cx50D47A increased levels of Trib3 and Irs2 transcripts, we performed RT-qPCR. Levels of both Trib3 and Irs2 were significantly increased in Cx50D47A homozygotes, whereas the values in heterozygotes were similar to those in wild type animals (Fig. 7, A and B). The increase was, on average, 1.3-fold in heterozygotes and 7.3-fold in homozygotes for Irs2 and 1.5-and 1000-fold for Trib3. Because Trib3 transcription is a direct target of CHOP, and TRIB3 inhibits the transcriptional activity of ATF4 and CHOP, we used immunoblotting to determine whether the levels of TRIB3 were increased. A TRIB3-immunoreactive band was detected in samples from Cx50D47A homozygous lenses, whereas TRIB3 was almost undetectable in samples from wild type and Cx50D47A heterozygous lenses (Fig. 7C). On average, levels of TRIB3 protein represented 83% (heterozygotes) and 881% (homozygotes) of the values in wild type lenses (Fig. 7C).

Discussion
Cx50D47A does not traffic properly and is retained intracellularly, mostly in the endoplasmic reticulum (1). In this study, we have shown that expression of this mutant induced ER stress in the lenses of both heterozygous and homozygous mutant mice. The induced ER stress was transduced through the PERK-dependent pathway. The extent of activation of this pathway was more pronounced in homozygotes than in heterozygotes. We did not detect activation of the other two signal transduction pathways; the degree of activation of the Ire1␣ pathway was unchanged, whereas the ATF6 pathway was significantly decreased.
A common characteristic of ER stress is the expansion of the endoplasmic reticulum. Such an expansion may be responsible for the 84 and 162% increases in calreticulin levels in heterozygous and homozygous Cx50D47A lenses. This may result from an increase in the capacity of the ER per cell or from an increase in the number of lens cells containing ER, because Cx50D47A lenses have organelle-containing cells reaching farther into the organ than do wild type lenses (3). The expansion did not increase all ER-resident proteins to the same extent. GRP78 levels increased by about 50% in both heterozygous and homozygous lenses. Protein-disulfide isomerase increased by about 13% in heterozygous and 21% in homozygous lenses (3). Moreover, the transcript levels for other ER-resident proteins (i.e. p58 IPK and ERP72) were unchanged. These results imply that expression of Cx50D47A caused expansion of the ER with selective changes among ER-resident proteins, as has been observed after induction of ER stress in other cell types (12,13).
The canonical ER stress-induced PERK signaling pathway is characterized by elevated phosphorylation of PERK, which increases phosphorylation of EIF2␣. Phosphorylation of EIF2␣ results in inhibition of general protein translation (14) and selective translation of ER chaperones and the transcription factor ATF4. ATF4 in turn increases transcription of several proteins, including the transcription factor CHOP (Fig. 8). In Cx50D47A mice, P-PERK immunostaining in heterozygous and homozygous lenses was significantly higher than in wild type lenses, suggesting activation of this ER stress signaling pathway. Although the increase in P-PERK immunostaining

Cx50D47A Causes ER Stress in the Lens
did not reach statistical significance between Cx50D47A heterozygotes and homozygotes, downstream targets exhibited differences suggesting that their activation was greater in homozygotes. Heterozygous Cx50D47A lenses showed smaller increases of the P-EIF2␣/EIF2␣ ratio and of Chop transcripts than homozygous Cx50D47A lenses and showed no increases of CHOP or ATF4 protein levels. Homozygous Cx50D47A lenses showed a significant increase in ATF4 levels associated with increases in Chop transcript and protein, suggesting activation of a UPR. These changes probably occurred in organellecontaining fiber cells that reach deeper into the lens in Cx50D47A mice (3).
Persistent activation of PERK signaling (as occurs in Cx50D47A lenses) has been associated with impaired cell proliferation and a global decrease in protein synthesis (15). Decreased cell proliferation and inhibition of general protein synthesis probably contribute to the decrease in lens size observed in heterozygous and homozygous Cx50D47A mice (3). The decrease in general protein translation may partially explain the severe reductions (Ͼ80%) in Cx50 levels in these mutant mice, but it cannot fully explain the decrease, because levels of other proteins (including Cx46) did not decrease to the same extent (3). Levels of selected proteins might be reduced by degradation through the ubiquitin-proteasome system or autophagy; ER stress or the UPR can interface with these pathways to preserve proteostasis (16). Thus, both inhibition of general protein synthesis and activation of selective protein degradation may contribute to the incomplete differentiation of fiber cells in Cx50D47A mice. Similarly, incomplete differentiation was observed in C2C12 myoblasts overexpressing the spliced form of Xbp1, since they did not generate mature myotubes when induced to differentiate (17).
Elevated PERK signaling can increase either cell survival or apoptosis, depending on the differential activation of downstream targets (15,18). Homozygous Cx50D47A lenses showed aspects of both kinds of responses. They exhibited increases in Chop transcripts and CHOP protein, which are most commonly associated with apoptosis. TUNEL staining (which detects DNA strand breaks) was increased and was most evident toward the center of the lens. Some epithelial or differentiating fiber cells may have undergone apoptosis, contributing to the smaller lens size. However, homozygous Cx50D47A lenses also showed increased levels of Trib3 and Irs2 transcripts, both of which have been identified as pro-survival factors in early stage erythroblasts when erythropoietin is limiting (11). Trib3 is a downstream target of CHOP and can inhibit the transcriptional activities of ATF4 and CHOP (19,20). TRIB3 has been ascribed pro-apoptotic effects in other systems. Thus, it may act as a switch between survival and apoptosis, with its pro-or anti-apoptotic function depending on cell type, context, and extent of activation. The ability of TRIB3 to regulate its own expression by inhibiting the transcriptional activity of ATF4 suggests that the fates of different cells may depend on the levels of TRIB3 protein attained in each cell. In the lens, TRIB3 (in combination with IRS2) may have acted as a pro-survival factor, because these lenses are already compromised due to the decrease in connexin levels, which would limit intercellular transfer of nutrients and signaling molecules to the center of the lens. Thus, the activities of IRS2 and TRIB3 acting as prosurvival factors may explain the lack of massive TUNEL staining in differentiating fibers (as would be expected from the increase in CHOP) and the absence of remarkable findings in hematoxylin-and eosin-stained Cx50D47A lens sections (2). ATF4 has previously been implicated in lens cell survival, because lenses from ATF4 knock-out mice undergo apoptotic cell death and ultimately degenerate (21). Thus, the consistent increase in ATF4 levels that we observed in 1-month-old homozygous Cx50D47A lenses may correspond to a compensatory change to avoid apoptotic cell death.
Our results obtained from Cx50D47A mice suggest that other connexin mutants may also contribute to pathology through ER stress and the UPR. Lenses of 2-month-old mice heterozygous for Cx50G22R or Cx50S50P contain increased levels of spliced Xbp-1 transcripts (without corresponding changes in protein), suggesting that the IRE1␣-dependent pathway may be activated (22). However, these results contrast with our observations that the IRE1␣-dependent pathway was not activated in Cx50D47A lenses; rather, the PERK-dependent pathway was stimulated. This implies that the ER stress induced by diverse Cx50 mutants may be transduced through different signaling pathways. ER stress-induced cell death through activation of the UPR has been reported for mammalian cells transfected with skin disease-linked Cx31 mutants (23); however, it remains to be determined whether ER stress contributes to the phenotype of connexin mutant-linked diseases other than cataracts in vivo.
ER stress and the UPR may have roles in cataracts caused by different etiologies. Previous studies have reported that ER stressors activate the UPR in lens epithelial cells, leading to increased levels of GRP78 (BIP) and ATF4 (24). Additionally, lens epithelial cells from galactosemic rats show increased levels of ATF4, GRP78, CHOP, and cell death (25). In these studies, the relative increase in levels of GRP78 was more pronounced than that of ATF4, whereas the opposite was true in Cx50D47A homozygous lenses. Similar to our data on Cx50D47A mice, an increase in ATF4 levels associated with a decrease in ATF6 levels was observed in homozygous ␣A-R49C-crystallin knock-in lenses (26). However, the homozygous ␣A crystallin mutant lenses show more severe changes than the Cx50D47A lenses, including a 5-fold increase in GRP78 levels, vacuoles, highly disorganized fiber cells, and increased epithelial and fiber cell death (26,27). The different magnitude of increase in GRP78 levels in this mouse model compared with Cx50D47A mice may be due to the different roles of Cx50 and of ␣-crystallins (which act as molecular chaperones) (28). Thus, the transducing pathways and the extent of activation of ER stress and UPR differ in cataracts of different etiologies. Moreover, the Cx50D47A mouse lenses have substantial compensatory mechanisms allowing cell survival and amelioration of cataract development.
Activation of UPR components contributes to differentiation programs of various cells, including those in the lens (29). Activation of the PERK-dependent pathway may contribute to decreased cell proliferation and differentiation of epithelial cells and to incomplete (or delayed) maturation of differentiating fiber cells. The severe reduction of Cx50 levels probably also contributes to the small lenses in Cx50D47A mice, because deletion of Cx50 (in Cx50-null homozygotes) results in decreased lens size (30,31) ascribed to reduced cell proliferation (32). Thus, activation of the PERK-dependent pathway together with Cx50 deficiency in the Cx50D47A mice may produce a more severe phenotype compared with that in Cx50-null mice (i.e. smaller lenses with nuclear cataracts in both Cx50D47A heterozygotes and homozygotes) (Fig. 8). Our results imply that expression of Cx50D47A disturbs the normal and controlled (i.e. constitutive) UPR activation, inducing a more persistent ER stress. We propose that a novel state of adaptation allows lens cell survival in the mutant mouse lenses.

Experimental Procedures
Chemicals-All chemicals were obtained from Sigma or Fisher unless otherwise specified.
Antibodies Animals-Cx50D47A (No2, ENU-326) mice (originally identified by Favor (33) by screening for the cataract phenotype following ethylnitrosourea mutagenesis) were maintained as described previously (3). All animal procedures followed the University of Chicago Animal Care and Use Committee guidelines.
Organ Culture-Lenses were dissected from 1-month-old wild type mice and placed in medium 199 without phenol red containing 1 M thapsigargin, 0.1 mM non-essential amino acids, 10% FBS, 2 mM glutamine, 100 units/ml penicillin G, and 10 g/ml streptomycin sulfate. After 24 h, lenses were collected and processed for RT-qPCR or immunofluorescence.
Reverse Transcription Real-time PCR-Lenses from 1-month-old mice littermates of different genotype were homogenized in QIAzol Lysis Reagent (Qiagen, Valencia, CA) using a glass-glass homogenizer, and the RNA was purified using the miRNeasy minikit (Qiagen). RNA concentrations were determined using a Nanodrop spectrophotometer (Nano-Drop Products, Wilmington, DE), and RNA quality was analyzed on an Agilent Bio-Analyzer (Agilent Technologies, Santa Clara, CA). cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) using a cDNA aliquot, a primer set (as indicated in Table 1), and Fast SYBR Green Master Mix (Life Technologies, Inc.). Primer sets were accepted for further work if their efficiencies were within a 95% confidence value and they did not produce primer dimers as assessed by the melting curves. All reaction mixes contained equal amounts of RNA. Each sample was run in triplicate for each primer set in any one of three independent experiments. The transcript for the housekeeping gene, cyclophilin A, was used to normalize the relative levels of RNA among the different genotypes. Graphs were prepared using SigmaPlot version 10.0 (Systat Software, Inc., San Jose, CA).
Immunoblotting-Whole lens homogenates from 1-monthold mice were prepared in PBS (pH 7.4) containing 4 mM EDTA, 2 mM PMSF, and cOmplete EDTA-free protease inhibitor mixture (Roche Applied Science) using a glass-glass homogenizer followed by sonication. Protein concentrations were determined in the total homogenates using the Bio-Rad Protein Assay Dye Reagent Concentrate based on the method of Bradford (34). Immunoblots for antibodies from Cell Signaling Technology were performed following the company's immunoblot protocol. Samples were collected in SDS-containing sample buffer and homogenized using a glass-glass homogenizer. Protein concentrations were determined using the Pierce 660-nm protein assay (Thermo Fisher Scientific). Aliquots from lens homogenates containing equal amounts of total protein were loaded in each lane and resolved on SDSpolyacrylamide gels. Gels were then blotted to Immobilon P membranes (Millipore, Bedford, MA) using a wet transfer apparatus, because we have found that this electroblotting system results in even transfer of the proteins across the lanes. (We confirmed equivalence of loading and transfer by staining of parallel gels with Coomassie Brilliant Blue or of membranes with Ponceau S. The predominant proteins stained in lens homogenates by these methods are crystallins. See supplemental Fig. 1.) Then the membranes were subjected to immunoblotting as described previously (35). The bands obtained in at least three independent experiments were quantified by densitometry using Adobe Photoshop CS3 (Adobe Systems Inc., San Jose, CA). The results are reported as relative values of the levels detected in wild type lenses. Graphs were prepared using SigmaPlot version 10.0 (Systat Software). Data were analyzed for statistical signifi-cance using Student's t test. A p value of Ͻ0.05 was considered significant.
Immunofluorescence-One-month-old lenses were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Then they were rinsed three times with PBS, transferred to 30% sucrose in PBS, and left at 4°C until they sank. Lenses were cryosectioned using an HM 550 M cryostat (Thermo Fisher Scientific), and sections were collected on polylysine-coated slides (PolySciences Inc., Warrington, PA). Then sections were incubated in PBS containing 5% normal goat serum and 0.3% Triton X-100 for 1 h at room temperature, followed by an overnight incubation in primary antibodies diluted in PBS containing 1% BSA and 0.3% Triton X-100 at 4°C. After several rinses in PBS, sections were incubated in secondary antibodies diluted in PBS containing 1% BSA and 0.3% Triton X-100 for 1.5 h at room temperature. Subsequently, sections were rinsed several times with PBS, incubated with DAPI (Life Technologies) for 15 min at room temperature to stain nuclei, and rinsed with PBS. Coverslips were mounted using 4% n-propylgallate in PBS/ glycerol (1:4) or ProLong Gold antifade mountant (Life Technologies). Specimens were observed with an Olympus Plan Apo N ϫ60 (numerical aperture 1.42) oil immersion objective using an Olympus IX81 (Center Valley, PA) confocal microscope using the laser company settings for Alexa488, Alexa594, and DAPI or with a Zeiss Plan Apochromat ϫ10 (numerical aperture 0.45) or ϫ40 (numerical aperture 1.0) objective in an Axioplan 2 microscope (Carl Zeiss Inc.) equipped with a mercury lamp and the appropriate filter sets. Figures were assembled using Adobe Photoshop CS3 (Adobe Systems) and show representative images from at least three independent experiments. P-PERK immunofluorescence was quantified using results of four independent experiments. Between 4 and 11 confocal images from each genotype were acquired from each experiment. The integrated density (⌺ (stained area ϫ mean intensity)) of immunopositive P-PERK particles in each image was quantified using ImageJ (36) after applying a threshold. The threshold was kept constant for all images within an experiment. The results are reported in arbitrary units as the average of the integrated density of all images in each individual experiment and as the overall mean for each genotype. Statistical analysis was performed using paired Student's t test. A p value of Ͻ0.05 was considered significant.
TUNEL Staining-Cryostat sections from paraformaldehyde-fixed lenses were subjected to TUNEL staining using the Fluorescein in Situ Cell Death Detection Kit (Roche Applied Science; 11684795910; lot 11905500), following the manufacturer's instructions after permeabilization of the sections in freshly prepared 0.1% Triton X-100, 0.1% sodium citrate for 5 min on ice. Sections treated with 96 Kunitz units/ml RNasefree DNase I (Qiagen) for 2 min were used as a positive control. Specimens were observed in an Axioplan 2 microscope (Carl Zeiss Inc.) equipped with a mercury lamp. Images were acquired with a Zeiss AxioCam digital camera using Zeiss Axio-Vision software. Figures were assembled using Adobe Photoshop CS3. The integrated density of TUNEL staining in each image was quantified using ImageJ after applying a threshold. The threshold was kept constant for all images within an exper-