In phagocytic cells, the endolysosomal system is a crucial regulator of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. Prime examples of this are retinal pigmented epithelial (RPE) cells, which are not only among the most actively phagocytic cells, continuously phagocytosing shed photoreceptor outer segments (OS), but also are post-mitotic cells having high metabolic activity and a high rate of autophagy. For proper RPE function, and to ensure the functional integrity of the neural retina, both phagocytosis and autophagy need to be in balance. We show that in the RPE, bA3/A1-crystallin is required for normal lysosomal-mediated waste removal. Our studies suggest that bA3/A1-crystallin acts via the AMPK-mTORC1 signaling pathway. Based on our findings, we postulate that loss of bA3/A1-crystallin inhibits these signaling pathways, leading to a defect in the V-ATPase-mediated acidification of the lysosomal machinery. Since bA3/A1-crystallin has been reported to be present in human drusen and a possible role for lysosomes in age-related macular degeneration (AMD) has been suggested, it is possible that perturbation of normal phagocytosis/autophagy in RPE could cause some manifestations of AMD.

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell-matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated. We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin. The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

PURPOSE:Occupational and environmental Pb-exposure is associated with protein aggregation diseases which typically present in elderly populations (Parkinsons and cataract). Post-translational processing of crystallins, the major structural proteins of the lens, is altered with short-term Pb-exposure in Fisher 344 rats. In addition, lenses from aged rats become opaque upon long-term exposure to Pb in organ culture. To explore the route to lens opacification in the presence of Pb, cultured lenses from young rats which exhibit higher metabolic activity in lens culture and are more susceptible to experimental cataract in vivo and in vitro were exposed to Pb and evaluated for morphological and biochemical alterations.METHODS:Following culture in Pb (as lead nitrate) for four days (in the presence/absence of oxidative challenge), lenses were examined for clarity, integrity of epithelial layer, and molecular stability including crystallin post-translational modification and choline transport. Clarity of lenses cultured with/without Pb for up to 8 days was assessed to determine if Pb exposure would accelerate opacification.RESULTS:Lenses cultured in Pb for four days exhibited epithelial abnormalities including epithelial cell multilayering and nuclei abnormalities with extension of the nucleated epithelial cells past the bow region. Alterations in crystallin post-translational modifications and decreased membrane transport of choline were noted without corresponding lens opacification or altered α-crystallin chaperone activity. Lenses treated with Pb according to the same exposure protocol with subsequent challenge by hydrogen peroxide became opaque while the contralateral control lenses did not. Lenses which were cultured in the presence of Pb for longer periods with no subsequent oxidative insult exhibited lens failure at earlier time points than did the controls.CONCLUSIONS:These data indicate that Pb-exposure can accelerate the degradation of the cultured lens through induction of epithelial cell abnormalities, induce structural protein modifications before opacity, and predispose the lens to opacification with subsequent oxidant challenge.

We previously reported that zebrafish alphaB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish alphaB-crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel alpha-crystallin transcript designated cryab2 and the resulting protein alphaB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human alphaB-crystallin and zebrafish alphaB1-crystallin, respectively. RT-PCR showed that alphaB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian alphaB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant alphaB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of alpha-lactalbumin and lysozyme. At 25 degrees C and 30 degrees C, zebrafish alphaB2 showed greater chaperone-like activity than human alphaB-crystallin, and at 35 degrees C and 40 degrees C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that alphaB2-crystallin makes up approximately 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different alphaB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish alphaB-crystallins perform divergent physiological roles. After gene duplication, zebrafish alphaB2 maintained the widespread protective role also found in mammalian alphaB-crystallin, while zebrafish alphaB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.

PURPOSE:Syneretic response to pressure variation of 1 atm or more has been demonstrated previously in bovine and human lenses with nuclear magnetic resonance (NMR) relaxation measurements. This study is designed to prove that a similar response is observable in smaller pressure increments closer to the normal physiological range.METHODS:Excised calf eyes were enucleated, the lenses dissected out, and immersed in medium. The lenses were exposed to incremental pressures ranging from 1 to 3 atm. At each pressure increment, a series of T1-weighted and spin echo images were acquired. Each image was segmented into seven regions of interest and tabulated. Longitudinal (T1) and transverse (T2) relaxation times were calculated from numerical fits of the experimental values to a one-term exponential expression.RESULTS:The relaxation parameters were obtained from fitting the image intensities to exponential equations, which yielded a relaxation time T and a pre-exponential factor M. The relaxation times and the pre-exponential terms of both parameters displayed linear dependence on pressure. There was no evidence of a threshold for syneretic response. Both T1 and T2 relaxation times decreased with increasing pressure. The pre-exponential term M2 increased with pressure, whereas M1 decreased with pressure; however, the slope of the latter was statistically not significantly different from zero.CONCLUSIONS:Syneretic response to pressure in calf lenses as determined by NMR relaxation measurements proved to be linearly dependent on pressure over a wide range of hydrostatic pressures. There was no range where the syneretic response was absent. These two findings allow the conclusion that the phenomenon observed hitherto at large pressure changes in vitro could be operative in vivo under physiological pressure changes during accommodation.

We have discovered a spontaneous mutation in the Sprague-Dawley rat with a novel eye phenotype that we have named Nuc1. The Nuc1 mutation behaves as a single semi-dominant locus with an intermediate phenotype in the heterozygotes. Heterozygotes exhibit nuclear cataracts. Homozygous Nuc1 rats are fully viable and have microphthalmia, retinal abnormalities and disruption of lens structure shortly before birth. The homozygous mutant shows no obvious pathology outside of the eye, indicating that the mutation is highly eye specific in its effects. An unusual feature of the mutation is that it prevents the normal programmed loss of nuclei from lens fiber cells, but does not affect the loss of other organelles. TUNEL, light, and electron microscopic studies show normal intact nuclei in lens fibers, in contrast to many other models with degenerate nuclei and unlike normal lenses where no such nuclei remain. The beaded filament protein, filensin, is down-regulated in fibers of Nuc1, while heat shock cognate 70 is up-regulated. Homozygous retinas are thicker than normal, and TUNEL labeling indicates roughly half the number of apoptotic cells compared to a wild-type retina. The transient layer of Chievitz persists in adult Nuc1 retina, indicative of delayed development. Hence, Nuc1 is a novel mutation that could be an eye-specific regulator of apoptosis.

Several studies have shown that macrophages play an active role in the initiation and completion of the programmed cell death process during development. Macrophages are called professional phagocytes, as their primary role is phagocytosis. The process of phagocytosis is complex and to date only poorly defined. It has also been postulated that macrophages around the developing lens likely migrate into the neural retina and differentiate into microglia after completion of their role as debris removers. We have identified ED1 immunopositive macrophages and CD11b/18 (OX-42) immunopositive macrophage-like cells in the vitreous chamber and sub-retinal space of a rat spontaneous mutation that we have termed Nuc1. The mutation appears to affect the programmed cell death process and is highly eye specific in its effects. While ED1 and ED2-immunopositive macrophages have previously been found surrounding the developing lens and are thought to play a role in the programmed regression of the tunica vasculosa lentis (part of the vascular structure present on the posterior surface of the lens during development), OX-42-immunopositive cells have not previously been identified in the vitreous chamber under normal or pathological conditions. Macrophage subpopulations surrounding the lens may differentiate into OX-42+ cells in Nuc1 following the release of lens material into the vitreous after the posterior capsule ruptures. In Nuc1 homozygotes, the posterior lens capsule ruptures before birth, causing lens material to be extruded into the vitreous compartment and damaging the tunica vasculosa lentis. Alternatively, OX-42+ cells may be recruited due to an inflammatory response both in the vitreous compartment and sub-retinal space. Inflammation is known to have an enhanced influx of phagocytic cells. Our data suggests that subpopulations of macrophages perform distinct functions in inducing apoptosis and phagocytic activity during normal conditions and in disease.

The retina serves as an excellent model in which to study vertebrate CNS development. We have discovered a spontaneous mutation in the Sprague–Dawley rat that results in a novel and unusual ocular phenotype, including retinal abnormalities, that we have named Nuc1. We have previously shown that the Nuc1 mutation appears to suppress programmed cell death in the developing retina. Here we report that maturation of both the retinal neurons and the retinal vessels is abnormal in Nuc1 homozygous rats. The developmental changes in the retinal neurons and vasculature are correlated with regard to degree of abnormality. As Nuc1 homozygotes mature, focal retinal detachment begins at approximately 3 months after birth, and near total traction retinal detachment, associated with pre-retinal fibrosis and neovascularization, is evident by 18 months. Electroretinographic studies at 2.5 months of age indicate that functional retinal degeneration precedes retinal detachment. The functional abnormality is most evident in rods and the inner retina, and is present in homozygous but not heterozygous mutants. Immunocytochemical studies of rod and cone photoreceptors indicate abnormalities in rod, but not cone, photoreceptors in Nuc1 homozygotes, consistent with the electroretinographic findings. In Nuc1 animals, the Muller cells are activated. Although such activation may result from inflammation, Muller cells in Nuc1 may be reacting to a neuronal influence. It appears that the Nuc1 mutation plays a regulatory role in both developing and maturing ocular tissues. The Nuc1 mutation may also serve as an important genetic tool to explore the relationships that may exist among gliosis, normal neuronal development, and normal vascular development and how abnormalities in these associations lead to common retinal diseases.

Cataract extraction is associated with the risk of posterior vitreous detachment, macular edema and retinal detachment possibly as a result of a disturbance to the vitreous body during surgery. While it is common for lens cortical fiber debris to leak into the vitreous humour during cataract extraction, the extent to which the vitreous humour is altered post-surgery is unknown. The current study examines the integrity of the vitreous humour of pseudophakic and phakic human donor eyes by comparing the proteome, the viscosity and the size distribution of macromolecules in different regions of the vitreous humour from human pseudophakic and phakic donor eyes. Major differences between the proteomes of anterior and posterior vitreous humour were observed in phakic and pseudophakic donor eyes. Seventeen spots identified as complete, modified or cleaved forms of alphaA-, alphaB-, betaA4-, betaB2, and gammaS-crystallins were present in the anterior vitreous humour of all pseudophakic eyes studied. Crystallins were not detected in the posterior vitreous humour of the pseudophakic eye or the vitreous humour of the phakic eye. Significant alterations in abundance and/or modification of transthyretin, alpha antitrypsin, and retinoic acid binding protein were observed in all locations of pseudophakic vitreous humour as compared to phakic samples. In addition, a significant decrease in the number and intensity of protein spots was observed for the posterior vitreous humour of pseudophakic eyes when compared to posterior vitreous humour of phakic eyes. Proteins which were affected include antioxidant proteins and enzymes such as carbonic anhydrase and trisephosphate isomerase. A reversal of the viscosity gradient, anterior to posterior, in the vitreous humour of pseudophakic eyes was observed concomitant with alterations in the distribution of 50 nm particles. These particles are likely primarily composed of hyaluronan. While varying degrees of vitreous degradation may have existed prior to surgery and may have contributed to the cataract formation, in no case did the phakic donor eyes exhibit the same alterations in the vitreous humour proteome, viscosity or particle sizes as did the pseudophakic donor eyes. The examination of phakic/pseudophakic donor eye pairs confirmed that the vitreous humour proteome and structural integrity were very similar in the matched phakic donor eye to eyes from donors with no history of cataract. Even though the number of samples for this study was limited, the observed changes support the hypothesis that alterations in the vitreous humour proteome occur in psuedophakic eyes with concurrent alterations in the structure of the vitreous humor. These modifications of the microenvironment of the retina may contribute to the development of retinal complications following cataract surgery.

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.

We demonstrate that expression of beta- and gamma-crystallins is associated with intraocular vessels during normal vascular development of the eye and also in the Nuc1 rat, a mutant in which the hyaloid vascular system fails to regress normally. Real-Time RT PCR, Western blot and metabolic labeling studies indicate an increased expression of beta- and gamma-crystallins in Nuc1 retina. The increased expression of crystallins was localized to the astrocytes surrounding the intraocular vessels. A similar pattern of crystallin expression was also observed in the retinal vessels during normal development. Cultured human astrocytes exposed to 3-nitropropionic acid, an established model of neuronal hypoxia, increased VEGF expression, as expected, but also increased expression of crystallins. Our data suggest that crystallins may function together with VEGF during vascular remodeling. Interestingly, in human PFV (persistent fetal vasculature) disease, where the hyaloid vasculature abnormally persists after birth, we show that astrocytes express both VEGF and crystallins.