Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different γD-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of αA-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that αA-crystallin may not be efficient to inhibit aggregation of lens γ-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.

UPR Activation and the Down-Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium.

PURPOSE: To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts. METHODS AND MATERIALS: The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6. RESULTS: In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control. CONCLUSION: These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract.

Knockout of αA-crystallin inhibits ocular neovascularization.

PURPOSE: The present study aimed to investigate the effects of αA-crystallin on pathologic ocular neovascularization. METHODS: Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and αA-crystallin-knockout (CRYAA[-/-]) mice were used for the in vivo study. αA-crystallin was knocked down in HUVECs by using a specific small interfering RNA (siRNA), and the effects of αA-crystallin knockdown on proliferation, migration, tube formation, and apoptosis were evaluated. Enzyme-linked immunosorbent assays were performed to investigate extracellular concentrations of vascular endothelial growth factor (VEGF). In vivo mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) were generated by using CRYAA(-/-) mice. The nonperfused area in the OIR model was measured in flat-mounted retinas, and angiogenesis resulting in CNV was evaluated in retinal sections. Western blot analysis was performed to investigate the phosphorylation status of vascular endothelial growth factor receptor 2 (VEGFR2, high affinity receptor for VEGF), AKT, PLCγ1, FAK, Src, p42/p44MAPK, p38MAPK, caspase-3, and caspase-9 in cultured HUVECs, as well as in the OIR and CNV models. RESULTS: The CRYAA siRNA not only induced HUVEC apoptosis but also inhibited exogenous and endogenous VEGF-induced cell activities, including proliferation, migration, and tube formation. Pathologic neovascularization was attenuated in the CRYAA(-/-) murine OIR and CNV models. Both in vitro and in vivo, the inhibition of angiogenesis was mediated by the suppression of VEGF secretion and the inhibition of the VEGFR2 signaling pathway; VEGFR2, AKT, PLCγ1, FAK, Src, p42/p44 MAPK, and p38 MAPK all showed reduced phosphorylation levels. In addition, CRYAA knockout led to increased levels of cleaved caspase-9 and caspase-3. CONCLUSIONS: Knockout of αA-crystallin inhibited pathologic neovascularization through the VEGF and VEGFR2 signaling pathways both in vitro and in vivo. These results suggest that αA-crystallin could be a novel pharmaceutical target for the prevention of ocular neovascularization.

Molecular mechanism of formation of cortical opacity in CRYAAN101D transgenic mice.

PURPOSE: The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. METHODS: RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RESULTS: RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis identified a series of terminal differentiation defects in N101D lenses, specifically, increased proliferation and decreased differentiation of lens epithelial cells (LEC) and decreased denucleation of lens fiber cells (LFC). The expression of Rho A was reduced in different-aged N101D lenses, and, conversely, Cdc42 and Rac1 expressions were increased in the N101D mutants. Moreover, earlier in development, the expression of major membrane-bound molecular transporter Na,K-ATPase was drastically reduced in N101D lenses. CONCLUSIONS: The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.

mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy.

Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development.

Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates.

BACKGROUND: alphaA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12-24 hours, compared to the expression of the endogenous Cryaa gene. RESULTS: Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact alphaA-crystallin 148 kb BAC (alphaA-BAC) and alphaA-BAC(DeltaDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing alphaA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of alphaA-crystallin locus derived from alphaA-BAC(DeltaDCR3), 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of alphaA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing alphaA-crystallin in the lens pit. CONCLUSION: We conclude that a 148 kb alphaA-BAC likely contains all of the regulatory regions required for alphaA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular alphaA-crystallin expression. Finally, deletion of DCR3 in either alphaA-BAC(DeltaDCR3) or Cryaa (15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.

Pb2+ exposure alters the lens alpha A-crystallin protein profile in vivo and induces cataract formation in lens organ culture.

Epidemiological data supports lead exposure as a risk factor for cataract development. Previous studies which demonstrated oxidative imbalances in the lens following in vivo Pb(2+) exposure support the idea that lead exposure can alter the lens biochemical homeostasis which may ultimately lead to loss of lens clarity with time. alpha-Crystallin, a major lens structural protein and molecular chaperone, undergoes various post-translational modifications upon aging which may contribute to decreased chaperone function and contribute to loss of lens clarity. This study evaluated the impact of 5 weeks of oral Pb(2+) exposure (peripheral Pb(2+) level approximately 30 microg/dL) on the alphaA-crystallin protein profile of the lens from Fisher 344 rats. Decreases in relative protein spot intensity of more acidic forms of alphaA- and betaA(4)-crystallin and of truncated forms of alphaA-crystallin were noted. This data indicates that changes in post-translational processing of crystallins do occur in vivo following short courses of clinically relevant Pb(2+)-exposure. In addition, organ culture of lenses from 4.5-month-old rats in 5 microM Pb(2+) resulted in opacities, demonstrating that lead is toxic to the lens and can induce a loss of lens clarity.

Modified alpha A crystallin in the retina: altered expression and truncation with aging.

Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.

[Transcription changes of alpha A-crystallins gene during the development of retinal degeneration in Rd, Rds and C3H mouse].

PURPOSE: To determine the relationship between retinal alpha A-crystallins and the development of retinal degeneration in rd, rds and C3H mice. METHODS: Total retinal mRNA was prepared from the retina of (rd) retinal degeneration, (rds) retinal degeneration slow, C3H and control C3B mice during the progress of retinal degeneration (Post-natal 3, 4, 5, 6, 8 weeks). The retinal expression of alpha A-crystallins was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. RESULTS: Expression of retinal alpha A-crystallins is rather stable in rd, rds and C3H mice during the progress of their retinal degeneration. The expression is also similar to that in normal C3B mice but different to that in Royal college of surgeons(RCS) rats. CONCLUSION: Retinal alpha A-crystallins may not involve in the course of RP in rd, rds and C3H mice. The molecular mechanism of RP in rd, rds and C3H may be different to that in RCS rat.