Stacey Hose

Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague-Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the betaA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate betaA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

Mutation in the βA3/A1-crystallin gene impairs phagosome degradation in the retinal pigmented epithelium of the rat

Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that βA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the βA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, βA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, βA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. βA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.

A potential role for β- and γ-crystallins in the vascular remodeling of the eye

We demonstrate that expression of β‐ and γ‐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 β‐ and γ‐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. Developmental Dynamics 234:36–47, 2005.

βA3/A1-Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis

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.

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 βA3/A1-crystallin disrupts both Notch signalling in astrocytes and formation of the astrocyte template. Here we show that loss of βA3/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 βA3/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 βA3/A1-crystallin is essential for normal endolysosomal acidification, and thereby, normal activation of Notch signalling in astrocytes.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36-47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

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 βA3/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.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Developmental abnormalities in the Nuc1 rat retina: A spontaneous mutation that affects neuronal and vascular remodeling and retinal function

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.