ßA3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.

Increased Lipocalin-2 in the retinal pigment epithelium of Cryba1 cKO mice is associated with a chronic inflammatory response.

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack ßA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.

Impaired endolysosomal function disrupts Notch signalling in optic nerve astrocytes.

Astrocytes migrate from the optic nerve into the inner retina, forming a template upon which retinal vessels develop. In the Nuc1 rat, mutation in the gene encoding ßA3/A1-crystallin disrupts both Notch signalling in astrocytes and formation of the astrocyte template. Here we show that loss of ßA3/A1-crystallin in astrocytes does not impede Notch ligand binding or extracellular cleavages. However, it affects vacuolar-type proton ATPase (V-ATPase) activity, thereby compromising acidification of the endolysosomal compartments, leading to reduced γ-secretase-mediated processing and release of the Notch intracellular domain (NICD). Lysosomal-mediated degradation of Notch is also impaired. These defects decrease the level of NICD in the nucleus, inhibiting the expression of Notch target genes. Overexpression of ßA3/A1-crystallin in those same astrocytes restored V-ATPase activity and normal endolysosomal acidification, thereby increasing the levels of γ-secretase to facilitate optimal Notch signalling. We postulate that ßA3/A1-crystallin is essential for normal endolysosomal acidification, and thereby, normal activation of Notch signalling in astrocytes.

Expression of ßA3/A1-crystallin in the developing and adult rat eye.

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat ßA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). ßA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, ßA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that ßA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of ßA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.

A spontaneous mutation affects programmed cell death during development of the rat eye.

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.

Geranylgeranyl pyrophosphate counteracts the cataractogenic effect of lovastatin on cultured rat lenses.

Statins are commonly prescribed cholesterol-lowering agents which inhibit the rate-limiting enzyme of the cholesterol biosynthetic pathway. In addition to inhibiting cholesterol synthesis, statins also inhibit the synthesis of other sterol and non-sterol compounds produced by the pathway including the isoprenoids, farnesyl (FP) and geranylgeranyl pyrophosphate (GGP). Certain proteins, most notably small GTP-binding proteins of the Ras superfamily, must be post-translationally modified by addition of a farnesyl or geranylgeranyl moiety in order to be properly targeted to membranes and to be active. Statins have been shown to affect cellular processes such as proliferation, signaling and apoptosis and it is likely that these effects are due, at least in part, to decreased isoprenoid synthesis. Certain statins have been shown to produce cataracts in experimental animals. We have previously demonstrated that lenses exposed to lovastatin during organ culture may develop cataracts as well, and we proposed that this resulted from decreased prenylation of small GTP-binding proteins. To test our hypothesis, rat lenses were exposed to lovastatin in organ culture with concomitant supplementation of the medium with GGP and/or FP. The results clearly demonstrated that GGP strongly inhibited lovastatin-induced lens opacification in this system while FP had little effect. GGP also markedly reduced the histological changes and the increased epithelial cell apoptosis induced in the cultured lenses by lovastatin. The data indicate that inhibition of protein prenylation, perhaps of Rho GTPases, is an important factor in the lovastatin-induced cataract in vitro.

Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells.

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.