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Mallika Valapala, Samuel J. Zigler, Jr., Stacey Hose, Gerard A. lutty, Debasish Sinha; A Mutation In βA3/A1 Crystallin Disrupts Astrocyte Template Formation And Retinal Vascularization. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4118.
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The primary vascular layer in the retina is intimately associated with the underlying meshwork of astrocytes that emerge from the optic nerve. Although, it has been suggested that the astrocyte template guides the endothelial cells to facilitate vessel growth, the molecular mechanisms underlying this process remain elusive. Here, we propose that Notch signaling is important for proper astrocyte template formation and maintenance of vascular homeostasis in the retina. We also report a novel role for βA3/A1-crystallin in regulating the activity of the Notch pathway
The function of βA3/A1-crystallin was studied by utilizing the Nuc1 spontaneous mutant rats in which βA3/A1-crystallin gene is mutated. Optic nerve astrocytes were isolated from the postnatal day 3-5 wild type and Nuc1 rats. Quantitative real time PCR and luciferase assays were used to measure the activity of the Notch pathway. The localization of Notch pathway proteins was studied using subcellular fractionation, confocal microscopy and transmission electron microscopy
In Nuc1 astrocytes, the expression of active Notch was greatly reduced with a concomitant reduction in the levels of Notch downstream proteins Hey and Hes and secretory Vascular endothelial growth factor (VEGF) compared to the wild type cells. The expression of total Notch1 receptor was however elevated in the Nuc1 cells. Our results show that neither the proteolytic processing of the Notch receptor nor the nuclear translocation of the active Notch was altered in the mutant astrocytes. Our data strongly suggests that a defect in the degradation of the Notch1 receptor leads to the inefficient removal of inactive Notch from the plasma membrane, resulting in impaired ligand-induced Notch signaling in the Nuc1 mutant astrocytes. We also show that βA3/A1-crystallin is localized to the lysosomes in optic nerve astrocytes and that the protein is important for the proper functioning of the lysosomal degradative pathway
Our studies provide evidence that impaired Notch signaling pathway could disrupt the normal cross talk between Notch and VEGF pathways. We also propose a novel function for βA3/A1-crystallin in regulating the crucial molecular pathways that mediate astrocyte-endothelial cell communication during vascular development in the retina
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